nihms573961.pdf

Phenazine redox cycling enhances anaerobic survival in

Pseudomonas aeruginosa

by facilitating generation of ATP and

a proton-motive force

Nathaniel R. Glasser

Suzanne E. Kern

, and

Dianne K. Newman

1,3

Divisions of Biology and Geological and Planetary Sciences, California Institute of Technology

Department of Biology, Massachusetts Institute of Technology

Howard Hughes Medical Institute

Summary

While many studies have explored the growth of

Pseudomonas aeruginosa

, comparatively few

have focused on its survival. Previously, we reported that endogenous phenazines support the

anaerobic survival of

P. aeruginosa

, yet the physiological mechanism underpinning survival was

unknown. Here, we demonstrate that phenazine redox cycling enables

P. aeruginosa

to oxidize

glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis

through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP

concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to

redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also

show that ATP hydrolysis is required to generate a proton-motive force using the ATP synthase

complex during fermentation. Together, our results suggest that phenazines enable maintenance of

the proton-motive force by promoting redox homeostasis and ATP synthesis. This work

demonstrates the more general principle that extracellular redox-active molecules, such as

phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy

generation.

Keywords

Pseudomonas aeruginosa

; survival; phenazines; anaerobic metabolism; pyruvate fermentation

Introduction

Pseudomonas aeruginosa

is an opportunistic pathogen that forms both acute and chronic

infections. It is well known to form biofilms in diverse environments, and microbial stasis

within biofilms is thought to complicate the treatment of many persistent infections. For

example, cells at the base of

P. aeruginosa

biofilms enter a dormant state with decreased

levels of transcription and translation, and they undergo physiological adaptations to

hypoxic conditions (Alvarez-Ortega and Harwood, 2007; Williamson

et al.

, 2012). These

Correspondence to: Dianne K. Newman.

Published as:

Mol Microbiol

. 2014 April ; 92(2): 399–412.

HHMI Author Manuscript

cells are substantially more resistant to antibiotics than actively growing cells (Williamson

et al.

, 2012). Moreover, nutrient deprivation triggers active responses for

P. aeruginosa

that

increase antibiotic resistance in growth-arrested cells (Nguyen

et al.

, 2011). Even under

active growth, many bacterial species, including

P. aeruginosa

, maintain a small population

of phenotypically resistant cells called persisters (Lewis, 2010). The persister state is

thought to be controlled by stochastic changes in cell physiology and metabolism (Keren

al.

, 2004; Lewis, 2010; Allison

et al.

, 2011). Together, these subpopulations of resistant

non-replicating cells can serve as reservoirs for recurrent infections (Lewis, 2010). Our

incomplete understanding of the metabolic changes underpinning microbial quiescence thus

limits our ability to treat chronic infections.

In addition to forming biofilms and persister cells during infections, pseudomonads

synthesize a class of redox-active pigments collectively termed phenazines (Price-Whelan

al.

, 2006; Mavrodi

et al.

, 2006). Phenazines are important virulence factors (Lau

et al.

2005) that serve as antibiotics towards microbial competitors (Baron and Rowe, 1981) and

damage mammalian cells (Britigan

et al.

, 1992). Phenazines can benefit

P. aeruginosa

serving as signaling molecules (Dietrich

et al.

, 2006), regulating persister cell formation

(Möker

et al.

, 2010), influencing colony morphology (Dietrich

et al.

, 2008; Dietrich

et al.

2013), and promoting iron acquisition and biofilm development (Wang

et al.

, 2011).

We previously reported that micromolar concentrations of phenazines can support anaerobic

survival by transferring electrons to an extracellular oxidant (Wang

et al.

, 2010). Although

the physiological basis for this survival mechanism was unknown, phenazine redox cycling

was correlated with a more oxidized intracellular environment for planktonic cultures (Price-

Whelan

et al.

, 2007; Sullivan

et al.

, 2011) and colony biofilms (Dietrich

et al.

, 2013),

suggesting that phenazines alter metabolism.

P. aeruginosa

is thought to form biofilms

within the lungs of cystic fibrosis patients (Singh

et al.

, 2000; Worlitzsch

et al.

, 2002), and

in many patients, the lung sputum contains phenazines at concentrations that can support

anaerobic survival (Wilson

et al.

, 1988; Hunter

et al.

, 2012; Wang

et al.

, 2010). Phenazines

are positively correlated with the progression of

P. aeruginosa

lung infections (Hunter

et al.

2012), and so they may directly alter the physiology of

P. aeruginosa

infections

in situ

Here, we sought to elucidate the physiological basis for phenazine-promoted anaerobic

survival in

P. aeruginosa

. We discovered that phenazines facilitate the conversion of

glucose and pyruvate into acetate. Our data support a model where phenazines dissipate

excess reducing equivalents, allowing for the coupling of acetate and ATP synthesis through

the activity of acetate kinase. ATP is required for survival, in part to maintain the proton-

motive force through the ATP synthase complex. Our results highlight the interplay between

redox homeostasis and ATP synthesis in the context of long-term survival, and illuminate

the important role extracellular redox active metabolites can play in facilitating core

metabolism.

Glasser et al.

Page 2

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

Results

Phenazine redox cycling requires the activity of acetate kinase to promote survival

Phenazine redox cycling occurs when a phenazine molecule undergoes alternating reduction

and oxidation reactions, resulting in the transfer of electrons from the reductant (

e.g.

NAD(P)H) to the oxidant (

e.g.

oxygen). Phenazines can cross cell membranes, enabling the

transfer of reducing equivalents from inside the cell to the extracellular environment. We

have previously shown that this transfer of reducing equivalents can promote anaerobic

survival (Wang

et al.

, 2010). We found that a carbon source, specifically glucose, is required

for anaerobic survival in the presence of oxidized phenazines (Wang

et al.

, 2010). To

understand how phenazines promote anaerobic survival, we began by exploring the role of

glucose.

To metabolize glucose,

P. aeruginosa

uses the Entner-Doudoroff pathway to convert one

glucose molecule into two pyruvate molecules (Figure 1a). This process is coupled to the

synthesis of one net ATP molecule and two excess reducing equivalents (NAD(P)H) (Figure

1a). Pyruvate can be further converted into acetate to produce additional ATP and reducing

equivalents (Figure 1b). To sustain anaerobic glycolysis,

P. aeruginosa

must regenerate the

oxidants (

i.e.

NAD

and NADP

) that were consumed during pyruvate and acetate

synthesis. We therefore hypothesized that phenazines, by dissipating excess reducing

equivalents, might promote the oxidation of glucose into acetate during anaerobic

glycolysis. In this model, acetate synthesis is coupled to ATP synthesis through the activity

of acetate kinase (Figure 1b), and it is the ATP synthesis that we expected to promote

survival.

To address this hypothesis, we tested mutants with markerless deletions of pyruvate

metabolism genes in a phenazine redox cycling survival assay. In this experiment, early

stationary phase cells were resuspended in anoxic MOPS-buffered minimal medium

containing glucose as the sole carbon and energy source. To control the concentration of

phenazines, we used strains with deletions in both phenazine biosynthetic operons (Δ

phzA1-

phzA2-G2

, abbreviated Δ

phz1/2

) (Dietrich

et al.

, 2006) and added phenazine-1-

carboxylic acid (PCA) back to the culture at a physiologically relevant concentration (75

μM) (Hunter

et al.

, 2012). We chose PCA because it is the biosynthetic precursor of all other

phenazines in

P. aeruginosa

and other pseudomonads (Mavrodi

et al.

, 2001; Mavrodi

et al.

2013), and also because it is synthesized whether oxygen is present or not. While oxygen

and ferric iron are likely oxidants of phenazines in infection environments (Wang and

Newman, 2008), we instead supplied an electrode poised at a potential sufficient to oxidize

phenazines in our culture vessels (

= +207 mV).

Consistent with the fact that

P. aeruginosa

is not known to ferment glucose, cultures

declined to about 0.0001% viability after seven days when provided with glucose alone

(Figure 2a). As previously reported (Wang

et al.

, 2010), the addition of phenazines and an

extracellular oxidizing potential improved anaerobic survival to 10% after seven days

(Figure 2a). A strain lacking lactate dehydrogenase, Δ

ldhA

, was not impaired in survival

relative to the Δ

phz1/2

parent strain (Figure 2a). However, strains lacking acetate kinase that

are unable to generate ATP by converting pyruvate into acetate, Δ

ackA

and Δ

ackA-pta

Glasser et al.

Page 3

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

failed to survive relative to the parent strain, with only about 0.001% of cells remaining

viable after seven days (Figure 2a).

We detected current through the extracellular electrode only when phenazines were present

(Supplemental Figure 1), consistent with the model that phenazines mediate electron transfer

from inside the cells to the electrode. After three days of survival, before we observed a loss

in viability (Figure 2a), the Δ

phz1/2

strain transferred a total charge of 11 C to the electrode.

Using the relationship

Q =

(

acv

), where

is the total charge transferred (11 C),

Faraday’s constant (96,485 C mole

−1

is the concentration of PCA (7.5 × 10

−5

M),

is the

reaction volume (0.1 L), and

is the average number of redox cycles, we calculated an

average of 8 redox cycles per molecule of PCA, a value similar to previous measurements

(Wang

et al.

, 2010).

To test if acetate is produced during phenazine redox cycling, we used high performance

liquid chromatography (HPLC) to directly measure the secreted metabolic products in our

experiment. We detected the production of acetate, succinate, and two additional unknown

products whose identification is a work in progress. Consistent with our hypothesis, cultures

with oxidized phenazines produced more acetate than did cultures without phenazines

(Figure 2b). After three days, in duplicate measurement, the Δ

phz1/2

strain produced 350

μM (5.9 × 10

−8

nanomoles CFU

−1

) and 330 μM (5.0 × 10

−8

nanomoles CFU

−1

) of acetate

with phenazine redox cycling but only 78 μM (1.3 × 10

−8

nanomoles CFU

−1

) and 130 μM

(2.1 × 10

−8

nanomoles CFU

−1

) of acetate without phenazine redox cycling. Succinate and

one unknown compound were produced in approximately equal amounts irrespective of

phenazines (Figure 2a), with the Δ

phz1/2

strain producing 2.4 × 10

−8

nanomoles CFU

−1

succinate after three days with oxidized phenazines. The other unknown product was

produced in greater amounts in cultures with oxidized phenazines than in cultures without

phenazines (Figure 2a).

We expected that acetate would be synthesized by acetate kinase (Figure 1b), which couples

acetate synthesis to ATP synthesis. To our surprise, the Δ

ackA

and Δ

ackA-pta

mutants

produced similar amounts of acetate as the parent strain during phenazine redox cycling with

glucose (Figure 2b), suggesting that acetate kinase is not the only active acetate-producing

enzyme in our experiment. The

P. aeruginosa

PA14 genome contains the gene

poxB

, which

codes for a putative pyruvate oxidase that converts pyruvate to acetate and CO

without

directly generating ATP or NADH (Chang and Cronan, 1983). Using quantitative reverse-

transcription PCR, we detected

poxB

transcripts during exponential growth, early stationary

phase, and after resuspension in anaerobic MOPS-buffered minimal medium with glucose

and 75 μM oxidized PCA. We also measured an increase in the relative abundance of

pyruvate oxidase transcripts to control gene transcripts (pyruvate dehydrogenase and the

housekeeping genes

clpX

and

recA

) after resuspension in minimal medium when compared

to exponential growth (Supplemental Figure 2). Pyruvate oxidase, encoded by

poxB

, may

therefore account for the observed acetate synthesis in the Δ

ackA

and Δ

ackA-pta

mutants

during phenazine redox cycling. Pyruvate oxidase may also account for some acetate

synthesis in the WT strain.

Glasser et al.

Page 4

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

Together, the survival defect of the Δ

ackA

and Δ

ackA-pta

mutants (Figure 2a) demonstrates

that the activity of acetate kinase is essential for long-term survival in our phenazine redox

cycling experiment. As the Δ

ackA

and Δ

ackA-pta

mutants still produce acetate (Figure 2b),

it is unlikely that acetate itself is sufficient for survival. Instead, our data are consistent with

a model where ATP synthesis by acetate kinase is required for anaerobic survival.

Phenazine redox cycling promotes ATP synthesis during pyruvate fermentation

In keeping with the fact that the Δ

phz1/2

ldhA

mutant survived as well as the parent strain

phz1/2

(Figure 2a), we did not observe lactate synthesis during phenazine redox cycling

with glucose (Figure 2b). This suggests that lactate dehydrogenase is not used to maintain

redox homeostasis during anaerobic glycolysis. In contrast, lactate dehydrogenase is

required for survival during pyruvate fermentation (Eschbach

et al.

, 2004). During pyruvate

fermentation,

P. aeruginosa

converts pyruvate into succinate, lactate, and acetate (Figure

1b). This process supports anaerobic survival but not growth (Eschbach

et al.

, 2004) and

uses acetate kinase to synthesize ATP (Figure 1b). If phenazine redox cycling is a general

mechanism for maintaining redox homeostasis, we predicted that phenazine redox cycling

should support redox homeostasis and ATP synthesis during pyruvate fermentation.

To test this prediction, we measured the anaerobic survival of the Δ

phz1/2

ldhA

strain

using pyruvate as the sole carbon and energy source. As expected, this strain was unable to

survive by fermenting pyruvate (Figure 3a). Consistent with our prediction, however, the

addition of PCA and an extracellular oxidizing potential improved the survival of the

phz1/2

ldhA

strain during pyruvate fermentation (Figure 3a).

In our model of survival, redox homeostasis allows for additional acetate and ATP synthesis

through the oxidative branch of the pyruvate fermentation pathway (Figure 1b). To further

confirm this model, we used HPLC to measure acetate synthesis during pyruvate

fermentation. The Δ

phz1/2

ldhA

strain produced more acetate when provided with

phenazines and an extracellular oxidizing potential (Figure 3b). We observed a difference in

acetate production before the Δ

phz1/2

ldhA

cultures began to lose viability (on day 3),

suggesting that the difference in acetate synthesis was not simply due to a difference in

survival.

To confirm that phenazine redox cycling provides an energetic benefit to

P. aeruginosa

, we

also measured the ATP concentrations of cultures with and without phenazine redox cycling.

Consistent with a coupling between acetate and ATP synthesis, Δ

phz1/2

ldhA

cultures with

oxidized phenazines maintained higher ATP concentrations than those without phenazines

(Figure 3c). This difference was again observed before the cultures began to lose viability,

suggesting that the difference in ATP concentration was not simply a consequence of cell

death.

ATP synthesis is limited by redox homeostasis during pyruvate fermentation

Our model suggests that, by helping to maintain redox homeostasis, phenazines promote the

conversion of pyruvate to acetate through the activity of acetate kinase. This model predicts

that redox homeostasis is the limiting factor for ATP synthesis during pyruvate

Glasser et al.

Page 5

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

fermentation. From the stoichiometry of pyruvate fermentation, acetate and lactate synthesis

are coupled through the common cofactor NAD(H) (Figure 1b). Sustained pyruvate

oxidation to acetate, and thus ATP synthesis, requires the regeneration of NADH to NAD

which can be accomplished by the activity of lactate dehydrogenase. To determine whether

this is indeed the case, we compared the concentration of relevant metabolites during

pyruvate fermentation for wild type (WT)

P. aeruginosa

PA14 and different mutant strains.

To initiate pyruvate fermentation, we cultured PA14 using pyruvate as the sole carbon

source aerobically, shifted the cells to an anoxic environment, and washed and resuspended

the cells in fresh anoxic medium. As expected, anaerobic WT cultures with pyruvate

survived for at least seven days, while cultures without pyruvate quickly declined in

viability (Figure 4a). In contrast to previous studies with PAO1 (Eschbach

et al.

, 2004;

Schreiber

et al.

, 2006), which demonstrated nearly complete survival, in our study we

observed only 50% survival after 7 days of anaerobic incubation with pyruvate.

As expected, mutants deficient in the conversion of pyruvate to acetate, Δ

ackA

and Δ

ackA-

pta

, displayed a survival defect during pyruvate fermentation (Figure 4a). A mutant deficient

in the conversion of pyruvate to lactate, Δ

ldhA

, had a survival defect similar to that of the

ackA

mutant (Figure 4a). To our surprise, a mutant unable to convert acetyl-CoA to acetyl-

phosphate, Δ

ackA-pta

, was more defective in survival than the Δ

ackA

mutant (Figure 4a).

The survival difference between the Δ

ackA

and Δ

ackA-pta

mutants might result from a

difference in intracellular acetyl-phosphate concentrations, as acetyl-phosphate can serve as

a phosphate donor in signaling pathways (Klein

et al.

, 2007). Alternatively, the Δ

ackA-pta

mutant might accumulate acetyl-CoA, starving other essential pathways that require

coenzyme A. Similar results have been observed in

E. coli

, where the expression of a

pathway to consume acetyl-CoA enhances the survival of a Δ

pta

mutant in stationary phase

(Chang

et al.

, 1999).

We used ion chromatography to confirm the biosynthesis of acetate, lactate, and succinate

from pyruvate as previously reported (Eschbach

et al.

, 2004) (Supplemental Figure 3). As

expected, both the Δ

ackA-pta

and Δ

ldhA

mutants produced significantly less acetate than the

WT strain (Supplemental Figure 3), suggesting that the Δ

ldhA

mutant is limited in its ability

to generate acetate and ATP using acetate kinase.

To confirm that the Δ

ldhA

strain is impaired in redox homeostasis and ATP synthesis, we

measured the NAD

, NADH, and ATP concentrations in cultures fermenting pyruvate. After

24 hours of anaerobic incubation with pyruvate as the sole carbon and energy source, both

the WT strain and ∆

ackA

mutant maintained an [NADH]/[NAD

] ratio of approximately 1

(Figure 4b). In contrast, the ∆

ldhA

mutant contained a more reduced intracellular

environment with an [NADH]/[NAD

] ratio of approximately 2 (Figure 4b). The Δ

ldhA

mutant also contained significantly less total NAD(H) than the WT strain and Δ

ackA

mutant

(Supplemental Figure 4). In support of our model, both the ∆

ldhA

and ∆

ackA

mutants

contained significantly less ATP than the WT strain (Figure 4c).

The reduced acetate synthesis and depleted ATP concentration in the ∆

ldhA

mutant suggest

that the ∆

ldhA

mutant is unable to synthesize ATP through acetate kinase. The elevated

Glasser et al.

Page 6

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

[NADH]/[NAD

] ratio suggests that acetate and ATP synthesis are limited by redox

homeostasis, corroborating the fact that phenazine redox cycling improves survival in the

∆

phz1/2

∆

ldhA

mutant during pyruvate fermentation (Figure 3a).

Arginine supports anaerobic ATP synthesis and survival independently of redox state

The survival defects of the Δ

ackA

and ∆

ldhA

mutants during pyruvate fermentation suggest

that ATP synthesis is an essential component of survival in

P. aeruginosa

. To test this

hypothesis using an independent anaerobic metabolism that produces ATP, we investigated

survival using arginine as the sole carbon and energy source. Arginine is used as an ATP

source during anaerobic growth through the arginine deiminase pathway (Vander Wauven

al.

, 1984) (Figure 1c). Previous work has shown that mutants in the arginine deiminase

pathway have reduced anaerobic survival in undefined complex media (Schreiber

et al.

2006), but to our knowledge there have been no reports of arginine alone serving as a

sufficient energy source for survival.

To determine whether arginine is sufficient for anaerobic survival, we cultured

aeruginosa

PA14 aerobically using arginine as the sole carbon source, shifted the cells to an

anoxic environment, and washed and resuspended the cells in fresh anoxic medium with

arginine. As with pyruvate, arginine alone was sufficient as an energy source to promote

anaerobic survival (Figure 5a). WT PA14 dropped to 30% viability after one day of

anaerobic incubation and slowly decreased in viability to 15% after seven days. In contrast,

cells incubated without arginine declined to 0.1% viability after seven days (Figure 5a). A

transposon insertion mutant in the arginine deiminase pathway,

arcC

::MAR2×T7 (Liberati

et al.

, 2006), rapidly declined in viability to 0.02% after seven days despite the presence of

arginine (Figure 5a). Similar results were obtained with a second independent transposon

insertion mutant,

arcA

::MAR2×T7, disrupting another gene in the same pathway (Figure 1c)

(data not shown). This demonstrates that arginine is sufficient to promote anaerobic survival

through the arginine deiminase pathway.

To confirm that disrupting the arginine deiminase pathway also disrupted ATP synthesis, we

measured NAD

, NADH, and ATP concentrations in cultures incubated with arginine. After

4 hours of anaerobic incubation with arginine as the sole carbon and energy source, both the

WT strain and

arcC

::MAR2×T7 mutant maintained similarly high [NADH]/[NAD

] ratios

of approximately 3 (Figure 5b). However, the

arcC

::MAR2×T7 mutant contained

significantly less ATP than the WT strain (Figure 5c), consistent with the disruption of the

ATP-generating step in the arginine deiminase pathway. The ability of the WT strain to

anaerobically generate ATP from arginine, despite an elevated [NADH]/[NAD

] ratio, is

consistent with the fact that the arginine deiminase pathway does not involve redox reactions

(Figure 1c).

Together, these measurements are consistent with the hypothesis that ATP synthesis, rather

than the [NADH]/[NAD+] ratio itself, is essential for long-term anaerobic survival in

aeruginosa

. Although redox homeostasis is essential when it is coupled to ATP synthesis, as

in glycolysis and pyruvate fermentation, survival correlates most directly with ATP

synthesis itself.

Glasser et al.

Page 7

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

ATP synthesis is required to maintain the proton-motive force using the F

-ATPase

complex during fermentation

The requirement for ATP suggests that

P. aeruginosa

consumes ATP during long-term

survival. During fermentation in many bacteria, the F

-ATPase complex, also known as

ATP synthase, hydrolyses ATP to extrude protons from the cytoplasm to the periplasm,

thereby generating a proton-motive force (PMF) (Krulwich

et al.

, 2011). Previous studies

have highlighted numerous reasons why a PMF might be required for survival; for example,

the PMF is required for the exchange of metabolites and other ions (Krulwich

et al.

, 2011),

the localization of cell division and cytoskeletal proteins (Strahl and Hamoen, 2010),

protonation of the peptidoglycan layer (Calamita

et al.

, 2001), and the regulation of

autolysis (Jolliffe

et al.

, 1981). We therefore hypothesized that

P. aeruginosa

requires ATP

to maintain its PMF during fermentation.

To test this hypothesis, we incubated pyruvate-fermenting cells anaerobically with

′

dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocation channel in the

subunit of ATP synthase (Sebald

et al.

, 1980). DCCD (dissolved in ethanol) was

efficient at killing anaerobic

P. aeruginosa

, reducing viability to 0.16% ± 0.07% after two

days (Figure 6a). Ethanol from the addition of DCCD did not significantly affect survival, as

a control culture with 0.2% ethanol maintained 88 ± 17% viability (Figure 6a). This suggests

that the activity of ATP synthase is required for survival during pyruvate fermentation in

aeruginosa

To confirm that the PMF is required for survival, we incubated cells with carbonyl cyanide

3-chlorophenylhydrazone (CCCP), an ionophore that equilibrates the proton concentration

across cell membranes and dissipates the PMF (Hopfer

et al.

, 1968). CCCP (dissolved in

ethanol) reduced the viability of anaerobic cultures to below 0.01% after two days (Figure

6a), confirming that the PMF is required for survival.

If the PMF is required for survival, then restoring the PMF should recover survival even in

the presence of DCCD. Although DCCD will inhibit oxidative phosphorylation, the cells

can still synthesize ATP through pyruvate fermentation. To recover the PMF after DCCD

treatment, we incubated cells anaerobically with nitrate, which serves as an alternate

terminal electron acceptor for the electron transport chain in

P. aeruginosa

(Carlson and

Ingraham, 1983; Van Alst

et al.

, 2009). Cultures incubated with pyruvate, DCCD, and

nitrate sustained 46% ± 7% viability after two days (Figure 6a), demonstrating that

respiration mitigates the killing effects of DCCD. Nitrate also improved the survival of

cultures that were depolarized with CCCP to 2% ± 1% (Figure 6a). We did not observe

significant growth after two days in cultures incubated with pyruvate, 0.2% ethanol, and

nitrate, although we did observe growth after three days (data not shown), suggesting a long

lag phase for growth by anaerobic nitrate respiration.

Because both pyruvate fermentation and the arginine deiminase pathway support anaerobic

ATP synthesis, we also tested the effects of DCCD and CCCP on cultures surviving

anaerobically with arginine. Cultures treated with 0.2% ethanol maintained 11% ± 1%

viability after two days (Figure 6b), consistent with the incomplete long-term survival we

observed with arginine (Figure 5a). As with survival on pyruvate, DCCD inhibited the

Glasser et al.

Page 8

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

survival of cultures incubated with arginine, reducing viability to 2.8% ± 0.2% (Figure 6b),

and 100 μM CCCP reduced viability to below 0.001% (Figure 6b). This suggests that

maintenance of the PMF by the F

-ATPase complex is a general survival strategy used by

P. aeruginosa

during fermentation.

To validate that DCCD caused death by preventing the generation of a PMF, rather than by

some non-specific mechanism, we used flow cytometry with the dye DiOC

(3) to assess the

membrane potential of DCCD-treated cultures. DiOC

(3) is a green-excitable dye that

accumulates intracellularly in polarized cells, where it undergoes a red fluorescence shift

that is indicative of a membrane potential (Novo

et al.

, 1999). Since this assay is not

quantitative in Gram-negative bacteria (Shapiro and Nebe-von-Caron, 2004), we assessed

the membrane potential qualitatively by comparing DCCD-treated samples to controls

depolarized with CCCP (for an example analysis, see Supplemental Figure 5). As expected,

pyruvate-fermenting cells maintained a detectable membrane potential. We observed a time-

dependent depolarization of the cells after treatment with DCCD (Figure 6c), while an

ethanol-treated control remained almost fully polarized (Figure 6c), demonstrating that

DCCD prevents maintenance of the PMF and induces depolarization of the cell membrane.

These results indicate that

P. aeruginosa

uses the ATP generated during fermentation to

maintain a PMF using the F

-ATPase complex, and that the PMF is required for long-

term anaerobic survival. To integrate our findings, we measured the membrane potential of

cells surviving anaerobically on glucose in our phenazine redox cycling experiment. After

three days of survival, 38% ± 6% of cells maintained a detectable membrane polarization in

the presence of oxidized phenazines, while less than 1% of cells maintained a detectable

membrane potential in the absence of phenazines. Together, our results suggest that the ATP

generated during phenazine redox cycling is used to maintain the PMF.

Discussion

In this study, we sought an explanation for how phenazines promote anaerobic survival in

aeruginosa

be it in planktonic (Wang

et al.

, 2010) or biofilm (Dietrich

et al.

, 2013) cultures.

We found that phenazines help

P. aeruginosa

convert glucose and pyruvate into acetate

(Figure 2b, 3b). This was an interesting result because

P. aeruginosa

is not known to

ferment glucose or other sugars (Barnishan and Ayers, 1979; Eschbach

et al.

, 2004).

Previous studies have shown that small molecule electron carriers can couple fermentation

to extracellular oxidants, leading to the synthesis of more oxidized fermentation products

(Emde

et al.

, 1989; Emde and Schink, 1990; Benz

et al.

, 1998; Beck and Schink, 1995). Our

results suggest that phenazines perform a similar role, enabling

P. aeruginosa

to generate

ATP by oxidizing glucose into acetate. This anaerobic glycolysis promotes survival but not

growth (Figure 2a), underscoring the importance of studying survival phenotypes

independently from growth phenotypes.

In Figure 7, we integrate the known survival pathways and processes in

P. aeruginosa

with a

focus on redox homeostasis and ATP synthesis. By comparing phenazine redox cycling,

pyruvate fermentation, and the arginine deiminase pathway, our combined results suggest

that ATP synthesis is a key feature of long-term survival. The ATP synthesized during

Glasser et al.

Page 9

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

fermentation and phenazine redox cycling is required, in part, to maintain the PMF using the

ATP synthase complex.

During pyruvate fermentation, pyruvate dehydrogenase converts NAD

to NADH in a

pathway that allows acetate kinase to synthesize ATP (Figure 1b). NAD

is regenerated by

reducing pyruvate to lactate or succinate (Figure 1b).

P. aeruginosa

produces significantly

more lactate than succinate during pyruvate fermentation (Supplemental Figure 3)

(Eschbach

et al.

, 2004), and so lactate synthesis by lactate dehydrogenase appears to be the

primary pathway for NAD

regeneration. This effectively couples the activities of acetate

kinase and lactate dehydrogenase through the NAD(H) pool. As a result, the Δ

ldhA

mutant

has an elevated [NADH]/[NAD

] ratio during pyruvate fermentation (Figure 4b) and is also

unable to maintain ATP levels as high as the WT strain (Figure 4c). Phenazine redox cycling

can serve as an alternate pathway for redox homeostasis in the Δ

ldhA

mutant and restore

ATP synthesis (Figure 3c) and survival (Figure 3a).

In contrast to pyruvate fermentation, ATP synthesis by the arginine deiminase pathway does

not involve redox reactions (Figure 1c). During anaerobic incubation with arginine, both the

WT strain and the

arcC

::MAR2×T7 mutant developed an [NADH]/[NAD

] ratio even

higher than the Δ

ldhA

mutant did during pyruvate fermentation (Figure 5b). Nonetheless, the

WT strain maintained ATP levels considerably higher than the

arcC

::MAR2×T7 mutant

(Figure 5c), demonstrating that the arginine deiminase pathway can proceed even with large

intracellular redox imbalances. Despite maintaining a high [NADH]/[NAD

] ratio, cultures

incubated with arginine were able to survive through the arginine deiminase pathway

(Figure 5a). Pyruvate and arginine may therefore offer alternative sources of ATP during

long-term anaerobic survival.

The requirement for ATP suggests that

P. aeruginosa

is not dormant during long-term

survival. The ATP synthase inhibitor DCCD killed anaerobic PA14 cultures that were

fermenting pyruvate or arginine (Figure 6a,b) and also depolarized cultures that were

fermenting pyruvate (Figure 6c), demonstrating that

P. aeruginosa

actively maintains its

PMF using the ATP synthase complex during fermentation. Previous studies have shown

that survival also requires transcription (Hu and Coates, 1999), translation (Reeve

et al.

1984), and proteolysis (Weichart

et al.

, 2003), all of which consume ATP. By promoting

ATP synthesis, phenazine redox cycling can therefore facilitate a variety of cellular

processes that are required for long-term survival.

We do not yet know how native phenazines such as PCA are reduced

in vivo

. Phenazines

oxidize NAD(P)H

in vitro

(Cox, 1986), and so PCA might directly oxidize NAD(P)H within

the cell. Redox cycling compounds such as phenazines are oxidants of flavins and iron-

sulfur clusters (Gu and Imlay, 2011), and so metabolic enzymes such as NADH

dehydrogenase, pyruvate dehydrogenase, or pyruvate oxidase might also reduce phenazines.

Synthetic electron shuttles such as anthraquinone-2,6-disulfonate and paraquat do not

support anaerobic survival by redox cycling because they are reduced slowly by

aeruginosa

(Wang

et al.

, 2010), suggesting that cells specifically reduce native phenazines.

Our preliminary studies indicate PCA reduction is enzymatically catalyzed, and we are

attempting to identify the proteins responsible for this activity.

Glasser et al.

Page 10

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

Consistent with our finding that phenazines can alter metabolism, WT

P. aeruginosa

cultures grown aerobically with glucose secrete pyruvate in the oxygen-limited late

stationary phase of growth, while phenazine-null mutant cultures do not (Price-Whelan

al.

, 2007). It was suggested that superoxide or pyocyanin radicals may inhibit pyruvate

dehydrogenase, leading to pyruvate accumulation in the culture supernatant (Price-Whelan

et al.

, 2007). Our results suggest an additional interpretation where glycolysis is limited by

the availability of intracellular NAD

. By promoting redox homeostasis, phenazines might

accelerate the conversion of glucose to pyruvate faster than pyruvate can be consumed.

After the consumption of glucose, the secreted pyruvate can be fermented or oxidized in the

tricarboxylic acid cycle, depending on the metabolic needs of the cell.

Together, this work underscores the importance of environmental metabolites that are not

always accounted for in metabolic pathways. Extracellular electron shuttles, such as

phenazines, may permit cells to utilize unexpected energy sources by alleviating redox

constraints on a pathway. An understanding of environmental parameters that interface with

metabolism is therefore essential to assess the true metabolic potential of some organisms.

Furthermore, a distinction must be drawn between growth and survival when considering the

environment. Numerous studies have demonstrated the metabolic versatility of

aeruginosa

(for example, (Behrends

et al.

, 2009; Stover

et al.

, 2000)), and here we have

shown how several anaerobic pathways might enable survival under clinically relevant

conditions. Characterizations of cystic fibrosis sputum indicate the presence of up to 100 μM

phenazines (Hunter

et al.

, 2012) and significant quantities of nitrate, arginine, and glucose

(Palmer

et al.

, 2007). Nitrate concentrations may be as high as 350 μM (Palmer

et al.

, 2005)

and oxygen levels vary widely (Worlitzsch

et al.

, 2002; Kolpen

et al.

, 2014), potentially

resulting in extended periods of slow growth or quiescence. Given the metabolic

heterogeneity of biofilms (Williamson

et al.

, 2012) and the likely heterogeneity of

infections,

P. aeruginosa

cells throughout infection environments might exhibit a range of

growth rates, including survival without replication. As a result, understanding the multitude

of survival mechanisms used by

P. aeruginosa

may be key to identifying new drug targets.

Materials and Methods

Bacterial strains and growth conditions

The strains and plasmids used in this study are shown in Supplemental Table 1. For routine

growth,

P. aeruginosa

and

E. coli

were cultured at 37 °C in lysogeny broth (LB) containing

10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and optionally 15 g/L agar for solid

medium.

S. cerevisiae

was cultured at 30 °C in YPD medium containing 10 g/L yeast

extract, 20 g/L peptone, 20 g/L d-glucose, and optionally 20 g/L agar for solid medium.

Specific experimental growth conditions are described where appropriate. For aerobic

growth, liquid cultures were shaken at 250 rpm.

Construction and verification of P. aeruginosa mutants

Unmarked deletions in

P. aeruginosa

PA14 were constructed using a modification of

previously described methods (Shanks

et al.

, 2006). Briefly, PCR was used to amplify

approximately 1-kilobase fragments flanking the target gene. Homologous recombination in

Glasser et al.

Page 11

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

yeast was used to assemble these fragments into the

sacB

-counterselectable suicide shuttle

vector pMQ30. The knockout vector was transformed into

E. coli

DH5

, and triparental

mating with DH5

and

E. coli

HB101/pRK2013 was used to conjugate the constructed

knockout vector into PA14; merodiploids containing the chromosomally integrated vector

were selected on cetrimide agar (HiMedia) containing 100 μg mL

−1

gentamicin sulfate.

Merodiploids were cultured overnight in nonselective LB, and resolved merodiploids were

then selected on LB agar plates with 10% sucrose. Potential deletion mutants were screened

using colony PCR with primers flanking the target gene, and clean deletions were confirmed

by DNA sequencing of the PCR product (Retrogen). Transposon insertions were verified

using colony PCR with primers flanking the annotated transposon position. Primer

sequences for constructing and confirming the genetic mutants used in this study are listed

in Supplemental Table 2.

Phenazine redox cycling survival

The assay for anaerobic survival enabled by phenazine redox cycling was performed as

previously described (Wang

et al.

, 2010). Briefly, strains of PA14 deleted in the core

phenazine biosynthesis operons (∆

phz1/2

) (Dietrich

et al.

, 2006) were grown overnight in

LB. The overnight cultures were used to inoculate 250 mL of LB in 1-L flasks to an OD

500

of 0.05. At early stationary phase (OD

500

of 2.8) the cells were pelleted, washed twice

aerobically in MOPS-buffered medium (100 mM 3-(N-morpholino)propanesulfonic acid

(MOPS), 3.5 μM FeSO

, 43 mM NaCl, 3.7 mM KH

, 9.3 mM NH

Cl at pH 7.2), and

resuspended to a concentrated OD

500

of 70 before being transferred to a nitrogen-only

atmosphere (MBraun Unilab glove box). There, 1 mL of concentrated cells was added to

sealed glass vessels containing 100 mL of anoxic MOPS-buffered medium amended with 75

μM phenazine-1-carboxylic acid and 20 mM d-glucose or 40 mM sodium pyruvate. To

oxidize PCA, a graphite rod (Alfa Aesar #14738) working electrode was poised at a

potential of +207 mV vs. NHE against a homemade platinum mesh counter electrode, which

was placed in buffer within an attached small chamber separated from the bulk solution by a

glass frit. Electrochemical parameters were maintained by a potentiostat (Gamry). An Ag/

AgCl electrode (BaSi #RE5B) was used as the reference electrode. Anaerobic cultures of

cells were stirred vigorously and maintained at 31°C for the duration of the experiment, with

daily sampling for colony forming units. Phenazine turnover was confirmed by the

accumulated charge over the duration of the experiment as previously described (Wang

al.

, 2010).

Pyruvate and arginine survival

For measuring anaerobic survival using pyruvate or arginine as a sole carbon source,

cultures were grown overnight at 37 °C in a minimal medium containing 14.15 mM

, 35.85 mM K

HPO

, 42.8 mM NaCl, 9.3 mM NH

Cl, 1 mM MgSO

, 7.2 μM

FeCl

and trace elements (Widdel

et al.

, 1983), and 40 mM sodium pyruvate or 40 mM

arginine (pH adjusted to 7.2 with NaOH or HCl). Overnight cultures were diluted at least

100-fold with fresh medium to an OD

500

of 0.01 and incubated at 37 °C in shaking flasks to

an OD

500

of 0.4. After centrifuging the culture for 15 min at 5000×g, the supernatant was

removed and the pellet was transferred to a glove chamber (Coy) containing an atmosphere

of 15% CO

, 80% N

, and 5% H

. The pellet was washed twice with fresh anoxic medium

Glasser et al.

Page 12

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

and then resuspended to a final OD

500

of approximately 0.4 in rubber-stoppered tubes. The

sealed tubes were incubated at 37 °C in the anaerobic chamber without shaking.

Viability counting

For measurements of colony forming units (CFU), 20 μL of each culture was sampled

anaerobically and then serially diluted in aerobic phosphate-buffered saline. Relevant

dilutions were plated in drips of 10 μL on LB agar plates and incubated aerobically for 24

hours at 37°C. The average of at least 4 pipetting replicates was taken as the CFU count for

a given CFU measurement.

Measurement of NAD

, NADH, and ATP

The method for measuring NAD

and NADH in this study was based on established

protocols (San

et al.

, 2002; Price-Whelan

et al.

, 2007). For each condition, two 1-mL

samples in 1.7 ml tubes were centrifuged for 1 min at maximum speed (16,000×g). The

supernatant was removed and 200 μL of either 0.2 M HCl (for NAD

extraction) or 0.2 M

NaOH (for NADH extraction) was added and mixed by vortexing. The samples were then

incubated for 10 minutes at 55 °C, cooled on ice, and partially neutralized drop-wise with

200 μL of 0.1 M NaOH (for NAD

) or 0.1 M HCl (for NADH). Cell debris was pelleted by

centrifugation (5 minutes at maximum speed) and 150 μL of the supernatant was transferred

to a fresh tube for immediate analysis. To assay the amount of NAD

or NADH in each

sample, 5 μL of sample was added to 90 μL of the reagent mix in a 96-well microtiter plate.

The reagent mix contained 2× bicine buffer (1.0 M, pH 8.0), 1× water, 1× 40 mM EDTA, 1×

100% ethanol, 1× 4.2 mM thiazolyl blue, and 2× 16.6mM phenazine ethosulfate. The 96-

well plate was then heated to 30 °C. To initiate the colorimetric assay, 5 μL of a 1 mg mL

−1

solution of alcohol dehydrogenase II (Sigma A3263) in 0.1 M bicine pH 8.0 was added to

each well. A BioTek Synergy 4 plate reader was used to incubate the plate at 30 °C and

record the absorbance of reduced thiazolyl blue at 570 nm every minute for 30 minutes. The

rate of increase in absorbance at 570 nm over time was used as the measure of NAD(H)

concentration. Unknown concentrations of NAD

and NADH were determined from the

slope and intercept of a standard curve derived from known concentrations of NADH.

For ATP measurement, 20 μL of culture was added to 180 μL of dimethyl sulfoxide

(DMSO) to quench and dissolve the cells. The sample was then diluted with 800 μL of 0.1

M HEPES (pH 7.5) and stored at −80 °C for up to 7 days. ATP was quantified by mixing 25

μL of sample with 25 μL of Promega BacTiter-Glo reagent in a 96-well opaque white

microtiter plate. The BacTiter-Glo reagent uses luciferase to produce luminescence in an

ATP-dependent manner. Total luminescence was measured at 30 °C in a BioTek Synergy 4

plate reader. In control experiments using pure ATP, DMSO had no effect on luminescence

at a final concentration of 9% (data not shown). Unknown concentrations of ATP were

determined from the slope and intercept of a standard curve derived from known

concentrations of ATP.

Assessment of membrane potential

The membrane potential was measured qualitatively using the dyes 3,3

′

diethyloxacarbocyanine iodide (DiOC

(3)) and TO-PRO-3. Cultures were diluted at least

Glasser et al.

Page 13

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

100-fold into a permeabilization buffer containing 100 mM Tris, 1 mM EDTA, and 80 mM

NaCl (pH adjusted to 7.4 using HCl). DiOC

(3) and TO-PRO-3 (both dissolved in DMSO)

were then added to a final concentration of 30 μM and 100 nM, respectively. For

depolarized controls, CCCP (dissolved in DMSO) was added to a final concentration of 15

μM. The final DMSO concentration did not exceed 2%. The samples were incubated for 2-5

minutes at room temperature and then analyzed on an Accuri C6 flow cytometer. DiOC

(3)

fluorescence was measured using excitation at 488 nm and emission at 530 ± 15 nm (FL1)

and 610 ± 10 nm (FL3). TO-PRO-3 fluorescence was measured using excitation at 640 nm

and emission at 675 ± 12.5 nm (FL4). Intact cells were gated on a log-scale scatter plot of

FL4 vs. FL2 (585 ± 20 nm) for further analysis. Cells with a membrane potential were

distinguished by an increased red/green (FL3/FL1) fluorescence ratio relative to depolarized

controls. For an example analysis, see Supplemental Figure 5.

High performance liquid chromatography

Metabolite samples were collected by centrifuging 550 μL of culture and passing the

supernatant through a 0.2 μm nylon centrifugal filter. Samples were stored at −80 °C until

analysis. Pyruvate, acetate, and succinate were quantified using an Agilent 1100 Series

HPLC. A G1312A binary pump was used to a draw a continuous flow of 8 mN H

at 0.6

mL/min through a G1322A degasser. Samples were injected using a G1313A autosampler,

and separation was effected using an Aminex HPX-87H column (Bio-Rad). Each sample

was analyzed using a G1315A diode array detector to measure the UV absorbance at 206 nm

referenced to 260 nm (bandwidth 16 nm) with a total run time of 25 min. Retention times for

analytes were validated with single species standards. For each analyte, standards ranging

from 0 to 40 mM were used to calibrate peak area against a known concentration. Data were

analyzed using the ChemStation software (Agilent).

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

This work was supported by the Howard Hughes Medical Institute (HHMI). DKN is an HHMI Investigator, and

NRG and SEK were both supported by NSF graduate research fellowships. This work was supported in part by the

National Research Service Award (T32GM07676) from the National Institute of General Medical Sciences. We

thank Nathan Dalleska and the Environmental Analysis Center (Caltech) for help with metabolite analyses, Ron

Grimm for help with electrodes, and Ian Booth and members of the Newman lab for helpful discussions. The

authors declare no conflicts of interest.

References

Allison KR, Brynildsen MP, Collins JJ. Metabolite-enabled eradication of bacterial persisters by

aminoglycosides. Nature. 2011; 473:216–220. [PubMed: 21562562]

Alvarez-Ortega C, Harwood CS. Responses of

seudomonas aeruginosa

to low oxygen indicate that

growth in the cystic fibrosis lung is by aerobic respiration. Mol Microbiol. 2007; 65:153–165.

[PubMed: 17581126]

Barnishan J, Ayers LW. Rapid identification of nonfermentative gram-negative rods by the Corning

N/F system. J Clin Microbiol. 1979; 9:239–243. [PubMed: 107190]

Glasser et al.

Page 14

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

Baron SS, Rowe JJ. Antibiotic action of pyocyanin. Antimicrob Agents Chemother. 1981; 20:814–

820. [PubMed: 6798928]

Beck S, Schink B. Acetate oxidation through a modified citric acid cycle in Propionibacterium

freundenreichii. Arch Microbiol. 1995; 163:182–187.

Behrends V, Ebbels TMD, Williams HD, Bundy JG. Time-resolved metabolic footprinting for

nonlinear modeling of bacterial substrate utilization. Appl Environ Microbiol. 2009; 75:2453–2463.

[PubMed: 19218401]

Benz M, Schink B, Brune A. Humic acid reduction by Propionibacterium freudenreichii and other

fermenting bacteria. Appl Environ Microbiol. 1998; 64:4507–4512. [PubMed: 9797315]

Britigan BE, Roeder TL, Rasmussen GT. Interaction of the

Pseudomonas aeruginosa

secretory

products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to

endothelial cells. J Clin Invest. 1992; 90:2187–2196. [PubMed: 1469082]

Calamita HG, Ehringer WD, Koch AL, Doyle RJ. Evidence that the cell wall of

Bacillus subtilis

protonated during respiration. Proc Nat Acad USA. 2001; 98:15260–15263.

Carlson CA, Ingraham JL. Comparison of denitrification by

Pseudomonas stutzeri

Pseudomonas

aeruginosa

and

Paracoccus denitrificans

. Appl Environ Microbiol. 1983; 45:1247–1253.

[PubMed: 6407395]

Chang D-E, Shin S, Rhee J-S, Pan J-G. Acetate Metabolism in a

pta

Mutant of

Escherichia coli

W3110: Importance of Maintaining Acetyl Coenzyme A Flux for Growth and Survival. J

Bacteriol. 1999; 181:6656–6663. [PubMed: 10542166]

Chang Y-Y, Cronan JE. Genetic and Biochemical Analyses of

Escherichia coli

Strains having a

Mutation in the Structural Gene (

poxB

) for Pyruvate Oxidase. J Bacteriol. 1983; 154:756–672.

[PubMed: 6341362]

Cox CD. Role of pyocyanin in the acquisition of iron from transferrin. Infect Immun. 1986; 52:263–

270. [PubMed: 2937736]

Dietrich LEP, Okegbe C, Price-Whelan A, Sakhtah H, Hunter RC, Newman DK. Bacterial Community

Morphogenesis Is Intimately Linked to the Intracellular Redox State. J Bacteriol. 2013; 195:1371–

1380. [PubMed: 23292774]

Dietrich LEP, Price-Whelan A, Petersen A, Whiteley M, Newman DK. The phenazine pyocyanin is a

terminal signalling factor in the quorum sensing network of

Pseudomonas aeruginosa

. Mol

Microbiol. 2006; 61:1308–1321. [PubMed: 16879411]

Dietrich LEP, Teal TK, Price-Whelan A, Newman DK. Redox-active antibiotics control gene

expression and community behavior in divergent bacteria. Science. 2008; 321:1203–1206.

[PubMed: 18755976]

Emde R, Schink B. Oxidation of glycerol, lactate, and propionate by Propionibacterium freudenreichii

in a poised-potential amperometric culture system. Arch Microbiol. 1990; 153:506–512.

Emde R, Swain A, Schink B. Anaerobic oxidation of glycerol by Escherichia coli in an amperometric

poised-potential culture system. Appl Microbiol Biot. 1989; 32:170–175.

Eschbach M, Schreiber K, Trunk K, Buer J, Jahn D, Schobert M. Long-term anaerobic survival of the

opportunistic pathogen

Pseudomonas aeruginosa

via pyruvate fermentation. J Bacteriol. 2004;

186:4596–4604. [PubMed: 15231792]

Gu M, Imlay JA. The SoxRS response of

Escherichia coli

is directly activated by redox-cycling drugs

rather than by superoxide. Molecular Microbiology. 2011; 79:1136–1150. [PubMed: 21226770]

Hopfer U, Lehninger AL, Thompson TE. Protonic conductance across phospholipid bilayer

membranes induced by uncoupling agents for oxidative phosphorylation. Proc Natl Acad Sci USA.

1968; 59:484–490. [PubMed: 5238978]

Hu Y, Coates AR. Transcription of the stationary-phase-associated hspX gene of Mycobacterium

tuberculosis is inversely related to synthesis of the 16-kilodalton protein. J Bacteriol. 1999;

181:1380–1387. [PubMed: 10049366]

Hunter RC, Klepac-Ceraj V, Lorenzi MM, Grotzinger H, Martin TR, Newman DK. Phenazine content

in the cystic fibrosis respiratory tract negatively correlates with lung function and microbial

complexity. Am J Respir Cell Mol Biol. 2012; 47:738–745. [PubMed: 22865623]

Jolliffe LK, Doyle RJ, Streips UN. The Energized Membrane and Cellular Autolysis in Bacillus

subtilis. Cell. 1981; 25:753–763. [PubMed: 6793239]

Glasser et al.

Page 15

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

Keren I, Shah D, Spoering A, Kaldalu N, Lewis K. Specialized persister cells and the mechanism of

multidrug tolerance in Escherichia coli. J Bacteriol. 2004; 186:8172–8180. [PubMed: 15576765]

Klein AH, Shulla A, Reimann SA, Keating DH, Wolfe AJ. The Intracellular Concentration of Acetyl

Phosphate in

Escherichia coli

Is Sufficient for Direct Phosphorylation of Two-Component

Response Regulators. J Bacteriol. 2007; 189:5574–5581. [PubMed: 17545286]

Kolpen M, Kühl M, Bjarnsholt T, Moser C, Hansen CR, Liengaard L, et al. Nitrous Oxide Production

in Sputum from Cystic Fibrosis Patiens with Chronic

Pseudomonas aeruginosa

Lung Infection.

PLOS ONE. 2014; 9:e84353. [PubMed: 24465406]

Krulwich TA, Sachs G, Padan E. Molecular aspects of bacterial pH sensing and homeostasis. Nature

Rev Microbiol. 2011; 9:330–343. [PubMed: 21464825]

Lau GW, Hassett DJ, Britigan BE. Modulation of lung epithelial functions by

Pseudomonas

aeruginosa

. Trends Microbiol. 2005; 13:389–397. [PubMed: 15951179]

Lewis K. Persister cells. Annu Rev Microbiol. 2010; 64:357–372. [PubMed: 20528688]

Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E, Wu G, et al. An ordered, nonredundant

library of

Pseudomonas aeruginosa

strain PA14 transposon insertion mutants. Proc Natl Acad Sci

USA. 2006; 103:2833–2838. [PubMed: 16477005]

Mavrodi DV, Blankenfeldt W, Thomashow LS. Phenazine compounds in fluorescent Pseudomonas

spp. biosynthesis and regulation. Annu Rev Phytopathol. 2006; 44:417–445. [PubMed: 16719720]

Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS. Functional analysis of

genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from

Pseudomonas aeruginosa

PAO1. J Bacteriol. 2001; 183:6454–6465. [PubMed: 11591691]

Mavrodi DV, Parejko JA, Mavrodi OV, Kwak Y-S, Weller DM, Blankenfeldt W, et al. Recent insights

into the diversity, frequency and ecological roles of phenazines in fluorescent

Pseudomonas

spp.

Environ Microbiol. 2013; 15:675–686. [PubMed: 22882648]

Möker N, Dean CR, Tao J.

Pseudomonas aeruginosa

increases formation of multidrug-tolerant

persister cells in response to quorum-sensing signaling molecules. J Bacteriol. 2010; 192:1946–

1955. [PubMed: 20097861]

Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O, Beer K, et al. Active starvation responses

mediate antibiotic tolerance in biofilms and nutrient-limited bacteria. Science. 2011; 334:982–986.

[PubMed: 22096200]

Novo D, Perlmutter NG, Hunt RH, Shapiro HM. Accurate flow cytometric membrane potential

measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique. Cytometry.

1999; 35:55–63. [PubMed: 10554181]

Palmer K, Mashburn L, Singh P. Cystic fibrosis sputum supports growth and cues key aspects of

Pseudomonas aeruginosa

physiology. J Bacteriol. 2005; 187:5267–5277. [PubMed: 16030221]

Palmer KL, Aye LM, Whiteley M. Nutritional cues control

Pseudomonas aeruginosa

multicellular

behavior in cystic fibrosis sputum. J Bacteriol. 2007; 189:8079–8087. [PubMed: 17873029]

Price-Whelan A, Dietrich LEP, Newman DK. Rethinking 'secondary' metabolism: physiological roles

for phenazine antibiotics. Nature Chem Biol. 2006; 2:71–78. [PubMed: 16421586]

Price-Whelan A, Dietrich LEP, Newman DK. Pyocyanin alters redox homeostasis and carbon flux

through central metabolic pathways in

Pseudomonas aeruginosa

PA14. J Bacteriol. 2007;

189:6372–6381. [PubMed: 17526704]

Reeve CA, Amy PS, Matin A. Role of protein synthesis in the survival of carbon-starved

Escherichia

coli

K-12. J Bacteriol. 1984; 160:1041–1046. [PubMed: 6389505]

San K-Y, Bennett GN, Berríos-Rivera SJ, Vadali RV, Yang Y-T, Horton E, et al. Metabolic

engineering through cofactor manipulation and its effects on metabolic flux redistribution in

Escherichia coli

. Metab Eng. 2002; 4:182–192. [PubMed: 12009797]

Schreiber K, Boes N, Eschbach M, Jaensch L, Wehland J, Bjarnsholt T, et al. Anaerobic survival of

Pseudomonas aeruginosa

by pyruvate fermentation requires an Usp-type stress protein. J

Bacteriol. 2006; 188:659–668. [PubMed: 16385055]

Sebald W, Machleidt W, Wachter E.

'-dicyclohexylcarbodiimide binds specifically to a single

glutamyl residue of the proteolipid subunit of the mitochondrial adenosinetriphosphatases from

Neurospora crassa

and

Saccharomyces cerevisiae

. Proc Natl Acad Sci USA. 1980; 77:785–789.

[PubMed: 6444724]

Glasser et al.

Page 16

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript

Shanks RMQ, Caiazza NC, Hinsa SM, Toutain CM, O'Toole GA.

Saccharomyces cerevisiae

-based

molecular tool kit for manipulation of genes from gram-negative bacteria. Appl Environ

Microbiol. 2006; 72:5027–5036. [PubMed: 16820502]

Shapiro HM, Nebe-von-Caron G. Multiparameter flow cytometry of bacteria. 2004:33–44.

Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP. Quorum-sensing

signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature. 2000;

407:762–764. [PubMed: 11048725]

Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, et al. Complete genome

sequence of

Pseudomonas aeruginosa

PAO1, an opportunistic pathogen. Nature. 2000; 406:959–

964. [PubMed: 10984043]

Strahl H, Hamoen LW. Membrane potential is important for bacterial cell division. Proc Nat Acad

USA. 2010; 107:12281–12286.

Sullivan NL, Tzeranis DS, Wang Y, So PTC, Newman DK. Quantifying the dynamics of bacterial

secondary metabolites by spectral multiphoton microscopy. ACS Chem Biol. 2011; 6:893–899.

[PubMed: 21671613]

Van Alst NE, Sherrill LA, Iglewski BH, Haidaris CG. Compensatory periplasmic nitrate reductase

activity supports anaerobic growth of

Pseudomonas aeruginosa

PAO1 in the absence of

membrane nitrate reductase. Can J Microbiol. 2009; 55:1133–1144. [PubMed: 19935885]

Vander Wauven C, Piérard A, Kley-Raymann M, Haas D.

Pseudomonas aeruginosa

mutants affected

in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase

pathway. J Bacteriol. 1984; 160:928–934. [PubMed: 6438064]

Wang Y, Kern SE, Newman DK. Endogenous phenazine antibiotics promote anaerobic survival of

Pseudomonas aeruginosa

via extracellular electron transfer. J Bacteriol. 2010; 192:365–369.

[PubMed: 19880596]

Wang Y, Newman DK. Redox Reactions of Phenazine Antibiotics with Ferric (Hydr)oxides and

Molecular Oxygen. Environ Sci Technol. 2008; 42:2380–2386. [PubMed: 18504969]

Wang Y, Wilks JC, Danhorn T, Ramos I, Croal L, Newman DK. Phenazine-1-carboxylic acid

promotes bacterial biofilm development via ferrous iron acquisition. J Bacteriol. 2011; 193:3606–

3617. [PubMed: 21602354]

Weichart D, Querfurth N, Dreger M, Hengge-Aronis R. Global role for ClpP-containing proteases in

stationary-phase adaptation of

Escherichia coli

. J Bacteriol. 2003; 185:115–125. [PubMed:

12486047]

Widdel F, Kohring G-W, Mayer F. Studies on dissimilatory sulfate-reducing bacteria that decompose

fatty acids. Arch Microbiol. 1983; 134:286–294.

Williamson KS, Richards LA, Perez-Osorio AC, Pitts B, McInnerney K, Stewart PS, et al.

Heterogeneity in

Pseudomonas aeruginosa

biofilms includes expression of ribosome hibernation

factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the

metabolically active population. J Bacteriol. 2012; 194:2062–2073. [PubMed: 22343293]

Wilson R, Sykes DA, Watson D, Rutman A, Taylor GW, Cole PJ. Measurement of

Pseudomonas

aeruginosa

phenazine pigments in sputum and assessment of their contribution to sputum sol

toxicity for respiratory epithelium. Infect Immun. 1988; 56:2515–2517. [PubMed: 3137173]

Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al. Effects of reduced mucus

oxygen concentration in airway

Pseudomonas

infections of cystic fibrosis patients. J Clin Invest.

2002; 109:317–325. [PubMed: 11827991]

Glasser et al.

Page 17

Mol Microbiol

. Author manuscript; available in PMC 2015 April 01.

HHMI Author Manuscript