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Mmi 14132
Mmi 14132
14132
First published online 23 October 2018
molecule that eventually can kill the producing cells; after molecular oxygen, thereby generating superoxide, which
auto-poisoning, different cell death processes can be acti- by enzymatic or abiotic dismutation generates hydrogen
vated, which may or may not lead to cell lysis (breaking peroxide (Muller, 2002; Rada and Leto, 2013).
down of the cell membrane) and eDNA release (Rice and Even though PYO production can enhance biofilm
Bayles, 2008; Bayles, 2014). formation in P. aeruginosa (Ramos et al., 2010; Das et
In addition to being a model biofilm-forming organism, al., 2015), at which stages of biofilm development and
P. aeruginosa is also known for its production of color- precisely how it acts, are not fully understood. Previous
ful, redox-active molecules called phenazines – dibenzo studies suggest that PYO might mediate biofilm devel-
annulated pyrazines, containing two nitrogen atoms in the opment in at least three different complementary ways:
center ring that mediate proton-coupled electron transfer (i) causing auto-poisoning and eDNA release during early
(Laursen and Nielsen, 2004; Mavrodi et al., 2006; Price- stages of biofilm formation, mostly due to generation of
Whelan et al., 2006). Over the last decade, a variety of oxidative stress (Das and Manefield, 2012; Costa et al.,
beneficial physiological functions have been attributed to 2017), which we expand upon in this study; (ii) indirectly
phenazines, for example, cell signaling, electron shuttling modulating levels of the second messenger bis-(3′,5′)-cy-
to diverse oxidants and promotion of survival under elec- clic-dimeric-guanosine (c-di-GMP) and EPS production
tron acceptor limitation (Dietrich et al., 2006; Wang et al., (Okegbe et al., 2017); and (iii) stimulating expansion of
2010; 2011; Glasser et al., 2014), with different functions biofilms/aggregates under anoxic/hypoxic conditions,
attributed to different phenazines. One of the best stud- where PYO can function as an alternative electron accep-
ied phenazines is pyocyanin (PYO), which has medical tor and alleviate reducing stress (Dietrich et al., 2013;
importance and has long been considered a virulence Costa et al., 2017; Jo et al., 2017). In all cases, PYO-
factor that is toxic to host cells (Lau et al., 2004). This modulated effects—be they beneficial or deleterious, at
toxicity is mainly due to its capacity to quickly reduce the level of the single cell—seem to be tightly linked to
A B WT D Shrinking lysis
Explosive lysis
x 1.39 DIC
0.4
Proportion of dead cells
BEFORE
0.3
0.2
∆phz
0.1 TOTO
AFTER
0.0
∆phz WT
60 min 80 min
YFP
Fig. 1. Phenazine production increases cell death early in biofilm development, and cell death happens in two morphologically-distinct
ways.
A. Picture of a 6-hour WT biofilm showing dead cells stained with TOTO-1 iodide (magenta), which does not penetrate live cells; scale bar:
20 µm.
B. WT strain shows higher levels of cell death when compared to the ∆phz strain that cannot produce phenazines. Left: representative
pictures of attached live (green-DAPI) and dead (magenta-TOTO) cells – scale bars: 5 µm; right: quantification of cell death for WT and ∆phz.
Red lines show the average + standard deviation of the proportion of dead cells for both treatments. Ratio of cell death WT/∆phz = 1.39.
C. Time-lapse of cells growing on agar pads showing the two morphologically-distinct cell death events: ‘shrinking’ (blue circles) and
‘explosive’ lysis (magenta rectangles); scale bars: 5 µm. Zoomed in pictures for both are shown on the right (scale bars: 3 µm). Cells were
constitutively expressing yellow fluorescent protein, and fluorescence loss combined with morphology change (DIC) can be seen during cell
death.
D. Both explosive and shrinking lysis release DNA and other cellular contents into the environment. Explosive lysis quickly spreads eDNA
extracellularly (left), but eDNA also leaks out of dead cells after shrinkage events (right: note the cloud of fluorescence [white arrows] leaking
from the three dead cells [circles, DIC]). Scale bars: 5 µm. Brightness was increased for better visualization of the TOTO signal.
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
Pyocyanin’s effects on P. aeruginosa under different conditions 997
environmental and physiological conditions. Accordingly, production increases shrinking lysis during early bio-
we set out to determine how different conditions com- film development (Fig. 1B); to our knowledge, this cell
monly encountered by cellular populations at distinct death phenotype has not been previously characterized.
growth stages result in PYO-dependent cellular effects, It was previously shown that after explosive lysis, all the
focusing primarily on cell death. We demonstrate that cytoplasmic contents (including DNA) are expelled to
PYO production has predictable, distinct and dramatic the environment (Fig. 1D-left and Turnbull et al., 2016).
cellular consequences, according to the physiological To test if shrinking lysis events can also release eDNA
state of the producer. in the medium, we used TOTO-1 iodide, a nucleic acid
stain that does not penetrate living cells (Okshevsky and
Meyer, 2014). We observed that eDNA slowly leaks out
Results of shrunken cells, indicating that shrinking lysis provides
an alternate route for eDNA release at an early stage in
Phenazine production stimulates a morphologically
distinct type of cell death early in biofilm development biofilm development (Fig. 1D). Together, these data show
that phenazine production stimulates auto-poisoning in
We started by imaging early-stage biofilms attached to Pa PA14 biofilms, enhancing eDNA release to the envi-
glass, when cells settle on the surface and form micro- ronment by triggering a morphologically distinct type of
colonies. eDNA is known to play an important structural cell death.
role at this stage of biofilm development (Whitchurch et
al., 2002). We performed a standard biofilm development
assay and quantified phenazine auto-poisoning using the PYO increases cell death in liquid cultures under
wild-type (WT) strain of P. aeruginosa PA14 (Pa PA14) stationary phase conditions
that produces different phenazines, including PYO, and Heterogeneity during biofilm development and low effi-
a ∆phz strain that cannot make phenazines (both phz1 ciency of eDNA quantification in biofilm assays limit
and phz2 operons are deleted in this strain, see Dietrich the range of conditions we can readily control to study
et al., 2006). We grew WT and ∆phz Pa PA14 cells in phenazine-mediated eDNA release. Therefore, we
8-well chambers for 6 h. In this assay, cells attach to the switched to experiments using planktonic cultures. First,
coverslip on the bottom of the chamber to initiate bio- we checked whether the enhanced cell death we saw
film formation (Fig. 1A). We noticed significant amounts in our early stage biofilm experiments could be reca-
of cell death in these young biofilms (Fig. 1A). In addi- pitulated under planktonic growth conditions. We mea-
tion, we found that the WT attached cells died more than sured the number of colony forming units (CFU) from
∆phz cells (Fig. 1B), consistent with the observation that cultures of WT and ∆phz strains over the course of batch
phenazine production increases cell death in biofilms. growth (Fig. 2A). By the first time point (18 h), the WT
Next, we visualized cell death at the single-cell level. cultures had already produced considerable amounts
Because previous studies suggested that PYO produc- of phenazines in the medium, particularly the blue
tion can cause eDNA release (Das and Manefield, 2012), phenazine PYO (see tubes in Fig. 2A). Standard incuba-
we used this phenazine for these experiments. Cells in tor shaking (250 rpm) was sufficient to maintain PYO in
mid-exponential phase were washed and re-suspended its oxidized state (blue color). WT cultures consistently
on top of agarose pads containing 100 µM PYO and showed lower CFU recovery than ∆phz cultures, and
imaged using fluorescence microscopy. When looking this difference increased over time. Adding physiologi-
at the cells, we observed at least two morphologically cally relevant amounts of exogenous PYO (100 µM) to
distinct death events that we refer to as ‘explosive’ and the ∆phz culture recapitulated poisoning levels present
‘shrinking lysis’ (Fig. 1C). Explosive cell lysis in P. aeru- in WT (Fig. 2A).
ginosa occurs in different strains (including PA14, PAO1, In addition to PYO, P. aeruginosa strains produce and
PAK, PA103 and others), stimulating eDNA release and secrete other phenazines, including phenazine-1-car-
biofilm formation (Turnbull et al., 2016). It is caused by the boxylic acid (PCA), phenazine-1-carboxamide (PCN)
activation of a prophage endolysin (lys) present in the R- and 1-hydroxyphenazine (1-OH-PHZ) (Price-Whelan et
and F-pyocin gene cluster in the P. aeruginosa genome, al., 2006). Because different phenazines can affect cells
and is activated under stressful conditions (Turnbull et al., in different ways (Sakhtah et al., 2016), we compared
2016). We observed a very similar phenomenon happen- the toxicity of these four phenazines. ∆phz cells were re-
ing during our experiments (Fig. 1C). Yet, we also noticed suspended in phosphate buffer with a given phenazine, and
a different cell death phenotype, ‘shrinking lysis’, where survival was measured after 24 and 48 h. PYO is the most
cells contract in size at the moment of death (Fig. 1C); toxic phenazine (Fig. 2B), followed by 1-OH-PHZ, PCN, and
in contrast to explosive lysis, these cells remain visible PCA. Perhaps not surprisingly, phenazine toxicity under oxic
under the microscope after death (Fig. 1C). Phenazine conditions correlates with their oxygen reactivity: the faster
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
998 L. A. Meirelles and D. K. Newman
A C SUCCINATE GLUCOSE
n.s.
1e+10 ** n.s. Treatment
** ***
1.00 1.00
**
0.50 0.50
CFUs/mL
WT
OD500
n.s.
*** 0.25 0.25
1e+09 ***
Treatment
∆phz WT
∆phz
∆phz + PYO
1e+08 0.05 0.05
n.s.
∆phz
n.s. + PYO 0 10 20 30 0 10 20 30 40
n.s.
2500 2500
1e+07
2000 2000
PI (A.U.)
0 18 24 36
1500 1500
Time (hrs)
1000 1000
Reactivity with
B **
***
***
*** O2 (µM/h)
500 500
*** ***
PYO: 63.1 ± 11.2 0 0
100 *** ***
1−OH−PHZ: 36.5 ± 3.2 0 10 20 30 0 10 20 30 40
PCA: 14.4 ± 0.9
Time (hrs) Time (hrs)
Survival (%)
75
Treatment
No phenazines D 2000
PYO
1.00
50
1−OH−PHZ 0.50 1500
PI (A.U.)
OD500
PCN
PCA
0.25 1000
25
Treatment
No PYO 500
PYO (50 µM)
PYO (100 µM)
0 0.05 0
24 48 0 10 20 30 0 10 20 30
Time (hrs) Time (hrs) Time (hrs)
a phenazine reduces oxygen abiotically, the higher its tox- with high temporal resolution. To overcome this limitation,
icity (Fig. 2B) (Wang and Newman, 2008). Because (i) Pa we developed a high-throughput approach using a plate
PA14 produces significant amounts of PYO, (ii) PYO causes reader assay to quantify cell growth and death simultane-
the highest toxicity, and (iii) PYO production is known to be ously. Growth was measured by reading optical density
involved in eDNA release (Das and Manefield, 2012), we (OD500); at the same time, the fluorescence of propidium
focused specifically on PYO-mediated cell death and eDNA iodide (PI) dye added to cultures served as a proxy for cell
release for the remainder of our experiments. death. Similar to TOTO-1 iodide (used in the biofilm exper-
Though CFU counting can reveal the absolute num- iments), PI cannot penetrate live cells and thus has been
ber of viable cells in a culture for a given time point, this used as a cell death marker (Okshevsky and Meyer, 2014).
method is not conducive to sampling multiple conditions Because the TOTO fluorescence spectrum overlaps with
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
Pyocyanin’s effects on P. aeruginosa under different conditions 999
OD500
OD500
OD500
OD500
OD500
OD500
0.50
0.6 Treatment 0.6 0.6 0.6 0.6 Treatment
No PYO 0.25 4 mM 40 mM
PYO (100 µM) 10 mM 80 mM
0.4 0.4 0.4 0.4 0.4 0.10
20 mM
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
2000
1500 1500 1500 1500 1500
PI (A.U.)
1500
PI (A.U.)
PI (A.U.)
PI (A.U.)
PI (A.U.)
PI (A.U.)
1000 1000 1000 1000 1000 1000
500 500 500 500 500 500
0 0 0 0 0 0
0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50
Time (hrs) Time (hrs) Time (hrs) Time (hrs) Time (hrs) Time (hrs)
Fig. 3. Nutrient depletion triggers PYO-mediated cell death and PYO toxicity is coupled to the available reducing power.
A-E. Survival assays under different nutritional conditions. In all experiments, nitrogen was removed to minimize growth (see Experimental
Procedures). Starvation for carbon, phosphorus and sulfur causes increased PYO sensitivity.
F. Carbon starvation in stationary phase results in increased PYO-mediated cell death under normal growth conditions. Cell-death signal
increase correlates with carbon limitation, but higher levels of glucose (>40 mM) inhibit cell death. Concentrations for all other major nutrients
(N, P, S) were the standard used for our minimal medium (see Experimental Procedures). Plotted lines represent averages of six replicates
and shaded areas are SD. [Colour figure can be viewed at wileyonlinelibrary.com]
the background fluorescence of Pseudomonas cultures, (carbon, nitrogen, phosphorus, and sulfur) from the
PI was a better dye for this purpose (see Experimental medium on cells in the presence and absence of PYO.
Procedures). By measuring OD500 and PI fluorescence Cells were grown to mid-exponential phase, washed,
together, we could track the dynamics of auto-poisoning and re-suspended at high cell density in medium lack-
through PYO production at fine temporal resolution. ing each of these nutrients. The absence of nitrogen did
Confirming results from the CFU experiment, WT Pa not seem to impact the cells with respect to PYO sensi-
PA14 consistently had a higher cell death signal when tivity (Fig. 3A and Fig. S1). Therefore, we used nitrogen
compared to the ∆phz mutant (Fig. 2C). In addition, exog- removal as a way to limit growth (cells stopped growing
enously adding PYO to ∆phz increased the death signal ~5 h after the experiment started), enabling us to test
(Fig. 2C). The PYO-triggered cell death phenomenon is the importance of the other nutrients systematically (see
concentration dependent, with higher concentrations of Experimental Procedures). Removal of carbon, phos-
PYO promoting more cell death (Fig. 2D). The increased phorus or sulfur significantly stimulated PYO toxicity (Fig.
cell death of WT in comparison to ∆phz is evident regard- 3B–E), indicating that these nutrients might support PYO
less of the carbon-source (e.g. succinate or glucose – Fig. defense mechanisms under oxic conditions. To confirm
2C). The PI signal increase correlated with cells entering that cells were indeed dying, we performed viability tests
stationary phase (Fig. 2C). This was expected for the WT with CFU as a read out; this gave us confidence that the
strain, because PYO production is quorum-sensing reg- observed PI fluorescence increase could be interpreted
ulated (Sakhtah et al., 2013). However, PYO was exog- as cell death (Fig. S2). The absence of PYO always
enously added to ∆phz cultures at the beginning of the resulted in lower levels of cell death (Fig. 3).
experiment. Therefore, even when cells had 100 µM PYO Even though phosphorus and sulfur seem to be very
present in their environment prior to exponential growth, important for PYO defense, cells usually need significantly
cell death was triggered only after cells had entered sta- lower levels of these nutrients compared to carbon, with
tionary phase (Fig. 2C). This suggests the existence of S/C and P/C intracellular ratios in bacteria ranging from
specific physiological/environmental conditions during 0.02 to 0.08 and 0.04 to 0.09 respectively (Fagerbakke
stationary phase that cause PYO-mediated cell death. et al., 1996). Therefore, it is likely that carbon depletion
would occur first, leading us to speculate that its deple-
tion would be the central driver of the observed stationary
Nutrient depletion triggers PYO-mediated cell death
phase PYO-stimulated cell death. To test this hypothe-
Intrigued by the fact that PYO-triggered cell death only sis, we grew ∆phz cells under different concentrations of
occurred during stationary phase, we hypothesized that carbon (glucose) in the absence or presence of PYO to
a lack of specific nutrients might drive this phenotype. track poisoning temporally. A clear correlation between
Accordingly, we used our plate reader assay to test the glucose availability and cell death in the presence of PYO
effect of removing each of the main nutritional elements can be seen in Fig. 3F: lower levels of glucose resulted
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
1000 L. A. Meirelles and D. K. Newman
PI (A.U.)
OD500
after ~45 h, close to the end of the experiment. Further, 0.4 400
Treatment
increasing levels of glucose to 40 mM was sufficient to No PYO 200
completely inhibit PYO-mediated cell death (Fig. 3F). In 0.2 PYO (100 µM)
0
the absence of PYO, cell death was attenuated (Fig. S3), 0 10 20 30 40 50 0 10 20 30 40 50
indicating that under these conditions, PYO was primar- Time (hrs) Time (hrs)
ily responsible for cell death. Finally, we confirmed that
glucose in the medium was completely consumed before + Cells + Cells No Cells
B (reduction) (no reduction)
(reduction)
PYO-mediated cell death started by measuring bulk glu- 3.0
1200
cose levels in cultures at different time points (Fig. S4).
PYO (A.U.)
2.5
OD310
These data indicate that PYO’s toxic effects on P. aeru- 800
2.0
ginosa under oxic conditions are mitigated by available
400
carbon, the source of reducing power. 1.5
1.0 0
0 2 4 6 0 2 4 6 0 2 4 6
PYO also poisons cells under anoxic conditions Time (hrs) Time (hrs)
[ATP] µM
[ATP] µM
OD500
4 4 1.0 1.0 1.0
6
0.8 0.8 0.8
4
2 2 0.6 Treatment 0.6 0.6
2 No PYO
PYO (100 µM)
0 0 0 0.4 0.4 0.4
0h 8 18 26 8 18 26 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
n.s.
* * ** n.s. 2000 2000 2000
n.s.
100
PI (A.U.)
Survival (%)
Fig. 5. Reduced carbon and ATP synthesis are required to avoid poisoning caused by PYO.
A. ATP levels decrease if cells are in the presence of PYO under low carbon (glucose) concentrations (left-top); at the same time, a survival
drop can be detected, indicating poisoning and cell death (left-bottom). However, no decrease in ATP levels (right-top) or survival (right-
bottom) is detected if cells high levels of glucose are available. Differences were assessed using an unpaired t-test ([n.s. = p > 0.05 – not a
statistically significant difference]; [*p < 0.05]; [**p < 0.01]; [***p < 0.001]). Bar graphs represent averages of three replicates and error bars
are SD.
B. Inhibition of ATP synthesis using the F0F1-ATPase inhibitor DCCD (right) recapitulates the amount of cell death observed when cells are
incubated under low carbon concentrations (left). Significantly lower cell death is seen when cells can metabolize the available and abundant
reduced carbon (center). Cells survive well in all treatments when PYO is not present (beige lines). Plotted lines represent averages of six
replicates and shaded areas are SD. [Colour figure can be viewed at wileyonlinelibrary.com]
No substantial decrease in ATP levels or viability hap- ATP synthesis) and PYO toxicity, we sought to explore
pened to the control without PYO. On the contrary, for whether PYO induces particular defense mechanisms
cells incubated with high levels of glucose, the presence that require reduced carbon and ATP synthesis. We
of PYO did not elicit a drop in bulk ATP levels nor a sur- performed survival assays under conditions where cells
vival decrease (Fig. 5A-right). These data indicate that tolerate PYO well (i.e. oxic, nutrient-replete). After expos-
there is a consistent correlation between ATP depletion ing cells to PYO for 5 h, we measured the transcrip-
and PYO poisoning, with subsequent cell death. tional response of genes encoding different categories
To further test the necessity of ATP synthesis of energy-dependent defense systems. We separated
to avoid PYO poisoning, we exposed cells to N,N- the tested genes into four main categories: (i) oxidative
dicyclohexylcarbodiimide (DCCD), a chemical agent that stress response, (ii) efflux systems, (iii) SOS response
blocks the F0F1-ATP synthase and inhibits ATP synthe- and (iv) iron-sulfur (Fe-S) clusters biogenesis (Fig. 6A).
sis (Sebald et al., 1980), and measured their survival A strong induction of oxidative stress-related genes
under different conditions. We predicted that by inhibiting was observed after cells were exposed to PYO. These
ATP synthesis, DCCD would increase PYO toxicity even genes included alkyl hydroperoxide reductases (Ahps:
when abundant levels of glucose were available. Indeed, ahpC, ahpF, ahpB), a thioredoxin reductase (trxB2) and
addition of DCCD caused cells to die in the presence of a catalase (katB) (Fig. 6A). These genes are regulated
PYO even under high glucose concentrations, indicating by the transcription factor OxyR and induced by H2O2
that a lack of ATP sensitizes cells to PYO (Fig. 5B-right). (Ochsner et al., 2000; Hishinuma et al., 2008; Panmanee
Inhibiting ATP synthesis had a similar effect to limiting and Hassett, 2009). A thioredoxin gene (trxA) was also
cells for carbon (Fig. 5B-left), but when ATP synthe- mildly upregulated (Fig. 6). The catalytic activity of Ahps
sis was not inhibited, PYO poisoning was minimal (Fig. depends upon reducing power provided by reduced
5B-center). Together, these data indicate that ATP synthe- electron carriers such as nicotinamide adenine dinucle-
sis is necessary for cells to prevent PYO toxicity. otide (phosphate) (NAD(P)H) to perform the reduction of
H2O2 to water (Fig. 6B) (Imlay, 2013). Catalases on the
other hand, reduce H2O2 to water and O2 in a NAD(P)
Energy-dependent defense mechanisms are induced
H-independent manner and do not require stoichiomet-
when cells are exposed to PYO
ric reductants (Fig. 6B). That a strong oxidative stress
Having observed a strong correlation between nutri- response is part of the PYO defense mechanism under
tional conditions (particularly carbon metabolism and oxic conditions is not surprising (Hassan and Fridovich,
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
1002 L. A. Meirelles and D. K. Newman
A Fe-S cluster
Oxidative stress response Efflux systems SOS response biogenesis
8
Log2 Expression
Fold Change
4
−2
ahpC ahpF ahpB trxB2 katB trxA mexG mexH mexE mexF recA lexA PA14 lys iscA iscU fdx2
08300
B Ahp catalase
+ +
NADH + H + H2O2 2H2O + NAD 2H2O2 O2 + 2H2O
1980; Hassett et al., 1992; Rada and Leto, 2013). In (i) Fe-S cluster biogenesis machinery might participate in
addition, at least part of the oxidative stress response is the replacement/repair of damaged Fe-S clusters (Djaman
dependent upon reducing power. et al., 2004), and (ii) the Fe-S cluster biogenesis regulator
Besides the oxidative stress response, we suspected IscR contributes to resistance to oxidants (Romsang et al.,
other cellular processes would be upregulated by PYO 2014), we tested whether PYO induces genes involved in
and important for defense against it. For example, ener- Fe-S cluster biogenesis. In P. aeruginosa, the isc operon
gy-dependent RND efflux systems such a mexGHI-opmD (iron-sulfur-cluster formation) is the system for Fe-S clus-
are induced by PYO and involved in phenazine transport ter biogenesis (Romsang et al., 2014; Lee et al., 2015).
(Dietrich et al., 2006; Sakhtah et al., 2016). We tested We measured the induction of three different genes in this
two genes in this operon (mexG and mexH) and both of operon (iscA, iscU, fdx2) and found that all three genes
them were strongly induced by PYO in our survival exper- were upregulated in the presence of PYO (Fig. 6A).
iments (Fig. 6A). In addition, we tested another efflux
pump system, the mexEF-oprN operon, which is a multi-
A subpopulation of cells resists PYO poisoning even
drug efflux system in P. aeruginosa (Köhler et al., 1997);
under nutrient-limited conditions
its genes were also transcribed more in the presence of
PYO (Fig. 6A). We were also curious whether PYO expo- Together, our previous experiments indicated that contin-
sure would lead to DNA damage and a subsequent SOS uous ATP synthesis through oxidation of reduced carbon
response. Yet, the SOS-related genes recA and lexA helps cells avoid PYO auto-poisoning during the transi-
were only mildly induced (Fig. 6A). Moreover, we did not tion from a growth to non-growth state. However, these
detect PYO-upregulation of the endolysin lys, known to be experiments assessed the bulk response of populations,
responsible for the explosive lysis phenotype in P. aeru- leading us to wonder whether a subset of the population
ginosa (Turnbull et al., 2016) (Fig. 6A). This suggests that might respond differently. When exposing cells to PYO
the SOS response and ensuing lys-mediated explosive under stressful conditions (i.e. oxic, no nutrients), the
lysis are minimized when cells have sufficient nutrients to majority of the population dies (1-3 days after exposure),
power other defense mechanisms. but a small percentage of the population survives (usually
Finally, we were intrigued that sulfur seemed to signifi- around 5–15% for cells pre-grown in succinate, Fig. 2B).
cantly affect PYO resistance (Fig. 3D). Fe-S clusters are We were interested in understanding what enables this
ubiquitous prosthetic groups that allow proteins to perform subset of cells to resist PYO, and hypothesized that resis-
several functions, including electron transfer and disulfide tance could occur by two distinct means: (i) the surviv-
reduction (Johnson et al., 2005). PYO stimulates genera- ing cells are PYO resistant because they are in a distinct
tion of ROS (O2– and H2O2), and these species can dam- physiological state (i.e. ‘persister-like’) due to population
age Fe-S clusters (Imlay, 2006). However, phenazines can heterogeneity; or (ii) they could be surviving based on the
also directly oxidize Fe-S clusters in proteins (Gu and Imlay, nutrients released when the majority of the population
2011), which could potentially result in damage. Because dies.
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
Pyocyanin’s effects on P. aeruginosa under different conditions 1003
Survival (%)
(No PYO) (100µM) 75
Wash and 50
1st exposure
to PYO 25
0
No PYO PYO
Treatment
Survival (%)
(No PYO) (100µM) 125
Wash of survivors
100
and 2nd exposure 75
to PYO 50
25
0
No PYO PYO
Treatment
C Re-growth of “persister-like” cells and re-exposure to PYO - High toxicity
Survival (%)
Survivors after growth (No PYO) (100µM) 75
50
Wash and
Growth exposure to PYO
25
0
No PYO PYO
Treatment
Fig. 7. A subpopulation of cells can resist PYO even under nutrient-limited conditions. The experimental protocol is shown on the left with
results of survival after exposure to PYO on the right. Survival was quantified by plating and CFU counting. Differences were assessed
using an unpaired t-test. Bar graphs represent the average of three replicates and error bars are SD. [Colour figure can be viewed at
wileyonlinelibrary.com]
cultures. Despite these positive effects, phenazines are it begins to starve for certain nutrients (e.g. carbon). Even
more commonly considered to be toxic compounds that kill after cell death due to PYO auto-poisoning, an intrinsi-
competitors and damage host cells, and can even poison cally PYO-tolerant subset of the population still survives,
their producers (Lau et al., 2004; Voggu et al., 2006; Rada resulting in the opportunity for PYO to again become net
et al., 2008; Gibson et al., 2009; Das and Manefield, 2012; beneficial to the population when conditions alter. It is also
Wang et al., 2016). These opposing functions are striking important to consider that even though the toxic effects of
and paradoxical. The objective of this work was to gain a PYO can be detrimental at the level of the single cell, in
better understanding of how PYO’s ‘double edged sword’ the context of a biofilm, the ‘altruistic’ death of individuals’
effect impacts P. aeruginosa under particular conditions. permits eDNA release that provides a structural support
Our central hypothesis was that PYO’s versatile redox for the survivors. Accordingly, PYO’s nuanced ‘double
chemistry explains its orthogonal cellular effects in a pre- edged sword’ effect can be rationalized as net beneficial
dictable fashion, depending upon the environment and for the producer population over its lifecycle (Fig. 8).
physiological state of the cells. PYO is only produced at Critical to P. aeruginosa’s tolerance of PYO is its extensive
high cell density and when high levels of carbon are avail- genetic arsenal to cope with the consequences of PYO’s
able because it is regulated by quorum sensing (Dietrich redox activity (Hassett et al., 1992). This includes catalases,
et al., 2006). Under these conditions, when cells are oxi- Ahps, multiple thioredoxins and thioredoxins reductases
dant-limited, they can thrive by utilizing PYO as an elec- (Ochsner et al., 2000). Thioredoxins reduce disulfide bonds
tron acceptor and avoid toxicity because they are able in thiol groups that become oxidized due to oxidative stress
to power defense mechanisms. However, this benefit (Lu and Holmgren, 2014) and together with Ahps, many
to the overall population can quickly change, with PYO thioredoxin systems depend on the availability of reduced
becoming dangerous to the majority of the population as carbon provided by (NAD(P)H) (Lu and Holmgren, 2014).
Environment
- Low cell density
- Nutrient-independent survival of “persister-like”
- No PYO
subpopulation
- High nutrients
- Low cell density
- High oxygen levels
- When conditions are favorable, cycle re-starts
eDNA/nutrients release
Cellular response
- Redox homeostasis Cellular response
Environment Environment
- Defense mechanisms induced - Low levels of ATP/reducing power
- High cell density (oxidative stress, efflux pumps, - High cell density (defense mechanisms fail)
- High PYO Fe-S cluster biogenesis) - High PYO - PYO-derived cell death happens
- High nutrients - ATP/reducing power levels are - Low nutrients - Majority of the population dies
- Limited oxygen enough to sustain defenses
Fig. 8. Model of PYO’s ‘double-edged sword’ effect throughout the P. aeruginosa life cycle. At least four different growth stages where PYO
differentially affects P. aeruginosa can be distinguished and were tested by our experiments (A–D).
A. and B. Early stages where cells are resistant to PYO. Positive effect of PYO and the ability of cells to turn on energy-dependent
mechanisms that result in survival (see Figs 3A and F, 5 and 6).
C. Negative effects of PYO (poisoning and cell death) when nutritional conditions are unfavorable to power defense mechanisms (see Figs 1,
3B–F, 4 and 5).
D. Demonstration of the existence of a ‘persister-like’ subpopulation that is resistant to PYO (see Fig. 7). [Colour figure can be viewed at
wileyonlinelibrary.com]
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
Pyocyanin’s effects on P. aeruginosa under different conditions 1005
Because PYO is membrane permeable (Lau et al., 2004; Hall al., 2006) strains from an overnight culture were washed
et al., 2016), its containment (by chaperones and/or efficient and diluted (1:50) in succinate minimal medium (SMM,
transport systems) is also important to minimize unwanted composition: 40 mM sodium succinate, 50 mM KH2PO4/
cellular redox reactions, such as reactivity with Fe-S clus- K 2HPO4[pH 7], 42.8 mM NaCl, 1 mM MgSO4, 9.35 mM
ters; RND efflux systems powered by the proton-motive NH4Cl, trace elements solution (Widdel and Pfennig,
force are involved in this task (Piddock, 2006; Dietrich et 1981)), and grown until OD500 = 0.4–0.5. Cells were
al., 2006). Our results showing a requirement for reducing centrifuged, washed once, and diluted to OD500 = 0.25,
power (carbon) and sulfur to avoid PYO toxicity (Fig. 3), which was used as the final inoculum. Then, 250 µL of
together with the induction of genes involved in the oxidative WT or ∆phz inoculum were transferred to Nunc Lab-Tek
stress response, efflux pump and Fe-S cluster systems in 8-well chambered coverglass (Thermo Scientific) and
the presence of PYO (Fig. 6A), indicate that these defense incubated at 37°C in shaking conditions (250 rpm) during
systems are particularly important for PYO tolerance. Finally, six hours for biofilm development. This experiment was
though PYO can also intercalate into DNA (Hollstein and Van repeated 10 times, with two independent wells for WT
Gemert, 1971; Mavrodi et al., 2006; Das et al., 2015), in our and ∆phz cultures every time. After six hours of incuba-
experiments, cells seem to avoid extensive DNA damage tion, liquid was removed and the chambers were washed
(at least at the population level) when nutritional conditions twice with PBS (Phosphate buffer saline – 137 mM NaCl,
are adequate to support other energy-dependent defense 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.2)
mechanisms (only a mild SOS response was detected). to remove planktonic cells, leaving only the attached bio-
However, this does not exclude the possibility that an SOS films on the bottom of the chamber. Finally, biofilms were
response might be involved in auto-poisoning and lysis for a stained with both TOTO-1 Iodide (1 µM) and DAPI (20
subset of the population (Turnbull et al., 2016). Future stud- µM), and chambers were imaged using confocal micros-
ies using single-cell tracking will allow this to be determined. copy (Leica TCS SPE). For quantification of cell death of
Though we performed the majority of our experiments attached cells, 10 different fields of view per well were
in this report in planktonic culture for technical reasons, imaged. Images were then analyzed in Oufti (Paintdakhi
the implications for biofilm development are clear. Biofilms et al., 2016) using the DAPI channel for total cell counting
are heterogeneous and dynamic systems, and the ulti- (live and dead); when checked manually, the software
mate impact of PYO on the different subpopulations would was able to count 80–90% of the total cells for both WT
be expected to vary between beneficial and detrimental, and ∆phz treatments. Dead cells were manually counted
depending upon the microenvironment present within the using the TOTO channel and calculation of the propor-
biofilm at different stages of development. Parameters such tion of dead cells was done by dividing the number of
as nutrient concentrations, availability of electron acceptors dead cells/total number of cells (live and dead). The final
and the respective energetic state of the cells should dic- proportions of dead cells for each of the 10 replicates for
tate the effect that PYO (and other phenazines) will have WT and ∆phz are plotted in Fig. 1B.
on different regions of the biofilm. For example, under nutri- Single-cell experiments were performed using agarose
ent-replete conditions, PYO production can cause beneficial pads (SMM with 2% agarose). Cells were grown from an
expansion of biofilm aggregates under hypoxic/anoxic con- overnight culture until OD500 = 0.4–0.5, spotted on top
ditions (Costa et al., 2017). However, in regions of the biofilm of the pads and imaged using fluorescence microscopy
where cells are starved for nutrients and PYO is present, (Leica TCS SPE). Agarose pads contained 100 µM PYO
PYO-mediated auto-poisoning would contribute to eDNA and we used both WT and WT-YPF (constitutive YFP
release, an important structural component of the matrix. expression) strains for these experiments. Temperature
Finally, we note that fluctuations of nutrients, which occur in control was set to 37°C. Shrinking and explosive cell lysis
open systems (DeAngelis et al., 1989; Odum et al., 1995), were monitored in both Differential Interference Contrast
would be expected to significantly impact the effect of PYO. (DIC) and fluorescence channels under 100× magnifica-
These predictions are testable, and going forward, we hope tion. For eDNA staining, we used agarose pads contain-
to leverage the findings reported in this work to better under- ing TOTO-1 Iodide (1 µM).
stand how versatile, redox-active molecules such as PYO
and other phenazines can modulate biofilm development.
Liquid culture assays and quantification of phenazine
toxicity
Experimental procedures WT and ∆phz cells were grown from overnight cultures
to OD500 = 0.5 and then diluted into four independent
Biofilm and single-cell experiments
OD500 = 0.05 cultures (replicates) using 5 mL glass tubes
For biofilm experiments, cells of WT and ∆phz (both phz1 containing fresh SMM (with and without 100 µM PYO),
and phz2 operons are deleted in this strain, see Dietrich et where the experiment started. Cells were incubated at
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
1006 L. A. Meirelles and D. K. Newman
37°C for 36 h, and time-points were taken at 0, 18, 24 six wells with no PYO added, and six wells with 100 µM
and 36 h for plating and CFU counting. Plating for CFU PYO added), and the averages and standard deviations
counting was always done in lysogeny broth (LB) (Difco) for the six replicates were plotted. Each plate reader set
plates containing 1.5% agar. PYO final concentration up was repeated at least twice, with very similar results.
in WT cultures was quantified using high-performance For survival experiments under different nutritional
liquid chromatography (HPLC) (Fig. S6). For all experi- conditions presented in Fig. 3, ∆phz cells were grown in
ments shown in this paper, with the exception of the tox- GMM + 100 mM MOPS until OD500 = 0.7-0.8, centrifuged,
icity assay comparing different phenazines (see below), washed and re-suspended at OD500 = 2 in buffer (100 mM
PYO was dissolved in 20 mM HCl, which was always MOPS + 42.8 mM NaCl, pH 7). Then, the different nutrients
added to PYO-untreated cultures as a control. The PYO were added as indicated. Final concentrations for each
used was synthesized and purified using methods pre- nutrient were: carbon = 80 mM glucose; phosphorus = 50
viously described (Dietrich et al., 2006; Cheluvappa, mM KH2PO4/ K2HPO4 [pH 7]; sulfur = 1 mM MgSO4. In all
2014). Other phenazines were purchased from Princeton experiments, trace metals solution (Widdel and Pfennig,
Biomolecular Research Inc and TCI America. 1981) at the same concentration as the one used for growth
Toxicity of different phenazines was assessed by per- was added. During the survival experiments, the nitrogen
forming shock experiments where cells were shifted from source (NH4Cl) was omitted in all treatments as a way
growing conditions to minimal phosphate buffer (MPB, to minimize growth (OD500 still increased during the first
50 mM KH2PO4/ K2HPO4[pH 7], 42.8 mM NaCl) contain- ~5h of the experiment, but shortly afterward cells stopped
ing 100 µM of each of the four phenazines tested: PYO, growing). Removing nitrogen from the medium did not sen-
1-OH-PHZ, PCN and PCA. Cells were grown in SMM sitize the cells to PYO (Fig. 3A). To confirm that an increase
until OD500 = 0.7–0.8, washed, and concentrated to final in PI fluorescence indeed meant cell death, we plated cells
OD500 = 2 (which corresponds to ~2-3 × 109 cells) in MPB for CFU counting after 50 h of incubation (Fig. S2).
+ phenazines. In this experiment, phenazines needed to Finally, to further test the importance of carbon availabil-
be dissolved using a mutual solvent (DMSO), which was ity to survival against PYO, cells were grown in the pres-
used as a negative control. Incubation was done under ence or absence of PYO in GMM under different glucose
shaking conditions (250 rpm, 37°C) in 1.5 mL Eppendorf concentrations (4, 10, 20, 40 and 80 mM). For all survival
tubes containing 1 mL of culture. Cells were plated for CFU and growth experiments, OD500 measurements during
counting after 24 and 48 h of exposure to the phenazines. inoculum preparation were performed using a Beckman
Four replicates were performed for each treatment. Coulter DU 800 spectrophotometer. Measurements of pH
using pH strips after the end of the experiments revealed
no significant changes.
Plate reader assay for quantification of cell death
with a tungsten lamp using a red filter cube (excitation: described in Fig. 3A was performed, where cells were
486/20, emission: 620/40; settings available for the incubated under two treatments: (i) 100 µM PYO and
anaerobic plate reader). Direct comparison of absolute (ii) No PYO. Under these conditions, cells were able to
fluorescence values between aerobic and anaerobic survive and had nutrients necessary to power defense
measurements are not possible due to the different plate mechanisms against PYO toxicity. For all three repli-
readers used. Reduction of PYO by cells was monitored cates, 750 µL of culture containing ±PYO were split into
by two ways: (i) absorbance at OD310, which is intense five wells in a 96-well BRAND® microplate followed by
for oxidized PYO, but weak for reduced PYO (Wang and incubation in the plate reader for 5h. Cells were then
Newman, 2008); (ii) fluorescence at ~500nm using a harvested from the wells, pelleted and re-suspended
green filter cube (excitation: 360/40, emission: 460/40) in 215 µL of TE buffer (30 mM Tris.Cl, 1 mM EDTA, pH
which is intense for reduced PYO, but null for oxidized 8.0) containing 15 mg/mL of lysozyme + 15 µL of pro-
PYO (Wang and Newman, 2008). teinase K solution (20 mg/mL, Qiagen), and incubated
for 8–10 min. Further lysis steps and RNA extraction
were performed using an RNeasy kit (Qiagen) following
ATP measurements and ATP synthesis inhibition
the manufacturer’s instructions. For further removal of
ATP measurements were performed using the BacTiter- contaminant genome DNA, additional treatment of the
Glo reagent (Promega) in 96-well opaque white microti- extracted RNA with TURBO DNA-free kit (Invitrogen)
ter plates as previously described (Glasser et al., 2014). was performed following the manufacturer’s instructions.
In summary, aerobic survival experiments as described Finally, cDNA was synthesized from 1 µg of total RNA
above were set up under high or low carbon concentra- using iScript cDNA Synthesis kit (Bio-Rad) according to
tions (80 mM or 4 mM glucose, respectively). The expec- the manufacturer’s protocol.
tation was that cells under low carbon availability would For qRT-PCR, iTaq Universal SYBR Green Supermix
starve for carbon first, resulting in ATP depletion and cell (Bio-Rad) was used. Reactions were performed in 20 µL
death. After incubation, cultures were sampled at 8, 18 volume containing 10 µL of the supermix, 5 µL of cDNA
and 26 h for both ATP quantification and viability through and 5 µL of forward and reverse primer solution (2 µM
CFUs counting. For ATP measurements, 20 µL aliquot of each). Primers are listed in Table S1. Reactions were
each culture was added to 180 µL of DMSO. This solu- run using a 7500 Fast Real-Time PCR System machine
tion was diluted with 800 µL of 100 mM HEPES (pH 7.5) (Applied Biosystems). Standard curves for each primer
and stored at –80°C until analysis (no more than 2 days). pair using known concentrations of Pa PA14 genomic
Thawed samples were mixed 1:1 BacTiter-Glo reagent DNA were used to back calculate concentrations of cDNA
and luminescence was measured at 30°C using a plate for each gene of interest. Normalizations for a control gene
reader (BioTek Synergy 4). For determining ATP concen- were done using the house-keeping gene oprI (Babin et
trations in the sample, a standard curve with known con- al., 2016). Reactions with the additional gene proC were
centrations of ATP was generated. also run as a second negative control (Heussler et al.,
To test for a direct connection between ATP depletion 2015). After normalization by oprI, data is displayed as a
and PYO toxicity, experiments using the molecule N,N- log2 fold change in expression (Fig. 6A).
dicyclohexylcarbodiimide (DCCD) were performed. DCCD
is classical inhibitor of the F0F1-ATP synthase (Sebald
Testing a ‘persister-like’ phenotype upon PYO exposure
et al., 1980), disrupting ATP synthesis. Aerobic survival
experiments as described before were set up where cells To test if the survival of a subpopulation of cells upon
had three different treatments: (i) low carbon (4 mM glu- exposure to PYO under nutrient-limiting conditions hap-
cose) with no DCCD; (ii) high carbon (80 mM glucose) pens (i) due to release of nutrients from dead cells or (ii)
with no DCCD (i.e. normal ATP synthesis) or (iii) high car- due to a ‘persister-like’ phenotype, experiments followed
bon (80 mM glucose) + 200 µM DCCD (i.e. ATP inhibition). the scheme presented in Fig. 7. First, a ∆phz culture was
This concentration of the inhibitor was used because it grown in SMM as described for experiments measur-
did not seem to cause a negative impact on the negative ing phenazines toxicity, split in three different replicates,
control (No PYO treatment, Fig. 5B). OD500 and PI fluores- washed and shifted to MPB and exposed to 100 µM PYO
cence were measured over a time course of 40 h. for three days. The negative control did not contain PYO.
Then, survivor cells were pelleted and washed twice to
remove nutrients released from dead cells, followed by
RNA extraction and quantitative reverse transcriptase
re-suspension in MPB and a second exposure to PYO for
PCR (qRT-PCR)
three days. Finally, after second exposure to PYO, survi-
For RNA extraction, three independent cultures of ∆phz vors were grown again in SMM until OD500 = 1.0 and a
cells were grown in GMM and a survival experiment as third exposure to PYO (during six days) was performed
© 2018 John Wiley & Sons Ltd, Molecular Microbiology, 110, 995–1010
1008 L. A. Meirelles and D. K. Newman
to test if cells had become sensitive again. During all aeruginosa colony morphology mutants. Journal of
exposures to PYO, incubation was done under shaking Bacteriology, 184, 6481–6489.
conditions (250 rpm) at 37°C. PYO remained blue during Das, T., Kutty, S.K., Tavallaie, R., Ibugo, A.I., Panchompoo,
J., Sehar, S., et al. (2015) Phenazine virulence factor bind-
the entire experiment, indicating that oxygenation was
ing to extracellular DNA is important for Pseudomonas
sufficient to maintain the phenazine in its oxidized state. aeruginosa biofilm formation. Scientific Reports, 5, 8398.
Survival was assessed by plating and CFU counting. Das, T. and Manefield, M. (2012) Pyocyanin promotes extra-
cellular DNA release in Pseudomonas aeruginosa. PLoS
ONE, 7, e46718.
Statistical analyses DeAngelis, D.L., Mulholland, P.J., Palumbo, A.V., Steinman,
All statistical analyses were performed using R (R Core A.D., Huston, M.A. and Elwood, J.W. (1989) Nutrient dy-
namics and food-web stability. Annual Review of Ecology
Team, 2018). One-way ANOVA with post hoc Tukey’s HSD
and Systematics, 20, 71–95.
test for multiple comparisons was used in Fig. 2A and B. Dietrich, L.E.P., Okegbe, C., Price-Whelan, A., Sakhtah, H.,
Unpaired t-tests were used for data presented in Figs 5A Hunter, R.C. and Newman, D.K. (2013) Bacterial commu-
and 7. Before performing the mentioned statistical tests, nity morphogenesis is intimately linked to the intracellular
normality and homogeneity of variances were assessed redox state. Journal of Bacteriology, 195, 1371–1380.
using the Shapiro-Wilk test and Levene’s test, respectively. Dietrich, L.E.P., Price-Whelan, A., Petersen, A., Whiteley, M.
and Newman, D.K. (2006) The phenazine pyocyanin is a
terminal signalling factor in the quorum sensing network
of Pseudomonas aeruginosa. Molecular Microbiology, 61,
Acknowledgements 1308–1321.
Dietrich, L.E.P., Teal, T.K., Price-Whelan, A. and Newman,
We thank all the members of the Newman lab for exper- D.K. (2008) Redox-active antibiotics control gene ex-
imental advice and feedback on the manuscript; Megan pression and community behavior in divergent bacteria.
Bergkessel was particularly generous with her time Science, 321, 1203–1206.
and intellectual support. We also thank Scott Saunders Djaman, O., Outten, F.W. and Imlay, J.A. (2004) Repair of
for helping with pyocyanin quantification. Grants to oxidized iron-sulfur clusters in Escherichia coli. Journal of
Biological Chemistry, 279, 44590–44599.
D.K.N. from the ARO (W911NF-17-1-0024) and NIH 341
Fagerbakke, K.M., Heldal, M. and Norland, S. (1996)
(1R01AI127850-01A1) supported this research. Content of carbon, nitrogen, oxygen, sulfur and phos-
phorus in native aquatic and cultured bacteria. Aquatic
Microbial Ecology, 10, 15–27.
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