Bacterial Community Morphogenesis Is Intimately Linked to the
Intracellular Redox State
Lars E. P. Dietrich,a,bChinweike Okegbe,bAlexa Price-Whelan,a,cHassan Sakhtah,bRyan C. Hunter,a,dDianne K. Newmana,d
Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USAa; Department of Biological
Sciences, Columbia University, New York, New York, USAb; Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York,
USAc; Division of Biology, Division of Geological and Planetary Sciences, and Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California,
ing their architecture are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa produces phenazines, small
colony wrinkling. These results suggest that redox imbalance is a major factor driving the morphogenesis of P. aeruginosa bio-
on organisms that assume this lifestyle (1, 2). Multicellularity al-
lows division of labor, as well as protection from environmental
insults, but also presents a significant challenge by exacerbating
limitations for growth substrates. Eukaryotic macroorganisms al-
leviate this problem in part through (i) internal circulation that
allows delivery of substrates to specific locations and (ii) meta-
bolic differentiation. Although the significance of the multicellu-
lar lifestyle for metabolism and pathogenicity of microorganisms
munities to cope with substrate limitation are poorly defined.
Pseudomonas aeruginosa is a leading causative agent of noso-
comial infections that forms biofilms (i.e., surface-attached com-
It is also the primary cause of morbidity and mortality among
people with cystic fibrosis, in whom it aggregates within accumu-
lated mucus, causing chronic lung infections (6). The fact that P.
film and aggregate formation. Our research has focused on the
intricate structures formed by communities of P. aeruginosa as
they grow on the surfaces of rich media solidified with agar (“col-
phenazines are more rugose and start wrinkling earlier in the in-
cubation period (7) (Fig. 1A). Similar results have been obtained
for flow cell biofilms (8). The phenazines produced by P. aerugi-
nosa vary in structure and chemical properties (9, 10), but their
redox potentials are such that they all can be reduced by the bac-
terial cell and react extracellularly with higher-potential oxidants,
the bacterium and an external substrate (11).
In the early 20th century, E. S. Guzman Barron, Ernst Fried-
he ubiquity of multicellularity—a property observed in all
three domains of life—underscores the advantages conferred
heim, and others postulated that redox-cycling compounds such
as phenazines are “accessory respiratory pigments” that can sus-
tain bacterial “respiration” based on their ability to stimulate ox-
ygen consumption in suspensions of many different types of cells
(12–14). They speculated that these compounds can extend the
depth of respiration for cells deprived of oxygen, such as those
found in normal tissues and tumors (15, 16). This work was car-
ried out before respiratory pathways were fully understood and
well before the importance of microbial biofilms in nature and
disease was widely recognized. In this regard, Barron, Friedheim,
and their colleagues were both ahead of their time and handi-
capped by a lack of information. In the interval between these
pioneering studies and the present work, attention shifted to ex-
ploring the roles of phenazines as virulence factors (17). Over a
decade ago, we revived the “respiratory pigment” hypothesis in a
biofilm context, speculating that the capacity for extracellular
electron transfer might provide a physiological benefit for oxi-
dant-limited cells (9, 18). While it has long been appreciated that
biofilms are metabolically heterogeneous and that oxygen avail-
knowledge, no study has yet demonstrated that endogenous elec-
tron shuttles such as phenazines increase the habitability zone for
biofilm cells. Evidence in support of this hypothesis, until now,
beyond it to demonstrate that the intracellular redox state, not
Received 21 December 2012 Accepted 27 December 2012
Published ahead of print 4 January 2013
Address correspondence to Lars E. P. Dietrich, LDietrich@columbia.edu.
C.O., A.P.-W., and H.S. contributed equally to this work.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
April 2013 Volume 195 Number 7Journal of Bacteriologyp. 1371–1380 jb.asm.org
phenazines per se, correlates with colony morphological develop-
MATERIALS AND METHODS
lysogeny broth (LB) (22) in 12- by 100-mm tubes at 37°C with shaking at
250 rpm. Growth conditions for colonies are described below.
Construction of deletion and complementation plasmids. Un-
marked deletions were generated for the genes napA and narG in PA14
wild type; ?phz, phenazine-null mutant. The scale bar represents 1 cm, and the images were taken after 3 days of colony development. (B) Oxygen profiles in
colony biofilms. Oxygen concentrations were measured in P. aeruginosa colonies on day 3 as a function of microelectrode depth in the colony. (C) Oxygen
depletion (calculated as the initial slope, typically between 0- and 20-?m depth, of oxygen profiles over depth in the colony, as depicted in panel B) for the base
and a wrinkle (spoke) during colony development. Wrinkles appear in ?phz colonies 1 day earlier than in WT colonies. The error bars represent the standard
deviations of this measurement in the bases or wrinkles of 5 independent colonies. (D) Oxygen depletion slopes from ?phz colonies (representing data plotted
in panel C) shown as a function of wrinkle thickness.
Dietrich et al.
jb.asm.org Journal of Bacteriology
and the linearized allelic-replacement vector pMQ30 were assembled by
gap repair cloning using the yeast strain InvSc1 (23). The resulting dele-
tion plasmid was transformed into Escherichia coli BW29427 and mobi-
lized into PA14 using biparental conjugation. PA14 single recombinants
were selected on LB agar containing 100 ?g/ml gentamicin. Potential
napA or narG deletion mutants were generated by selecting for double
recombinants by identifying strains that grew in the presence of 10%
sucrose. Strains with properties of double recombination were further
analyzed by PCR for the desired deletion.
For construction of the nap operon complementation plasmid
pAPW1, primers were designed using the P. aeruginosa PA14 genome
sequence to anneal 490 bp upstream of napE and to the last 19 bases of
napC, yielding a PCR product including the napEFDABC operon and a
tion sites (HindIII and NheI) engineered within the primers. It was then
ligated into plasmid pMQ72 digested with the same restriction enzymes
and treated with calf intestinal phosphatase (Sigma). The resulting plas-
mid, pAPW1, contains the napEFDABC operon under the control of its
tryptone (Teknova) was autoclaved and cooled to 60°C before 20 ?g/ml
Coomassie blue (EMD) and 40 ?g/ml Congo red (EMD) were added.
Sixty milliliters of medium was poured per 10-cm-square plate (Simport;
D210-16) and allowed to dry with closed lids at room temperature for
For colony spotting and developmental studies, precultures were in-
h. Ten microliters of the preculture was spotted on plates and incubated
microscope (Keyence; VHX-1000).
For colony development at different oxygen concentrations (15%,
21%, and 40%), we incubated plates in C-Chambers (BioSpherix; C274).
Oxygen concentrations were regulated by mixing pure nitrogen and oxy-
gen (TechAir) using the gas controller ProOx P110 (BioSpherix). Each
chamber contained an open 10-cm round plate filled with 25 ml of water
to keep the chambers humid. Humidity was monitored using an iButton
Humidity Data Sensor (Maxim) and maintained at ?90%. For colony
growth under anoxic conditions, plates were stored in an anaerobic glove
box filled with 80% N2, 15% CO2, and 5% H2.
ered with 4% paraformaldehyde and allowed to fix for 10 min. The colo-
to fix for an additional 10 min. Following this, the colonies were trans-
ferred into a wash basket, and the fixative was removed by washing the
colonies three times in phosphate-buffered saline (PBS). Excess PBS was
4583) in PBS before the colonies were transferred to disposable embed-
ding molds (Electron Microscopy Sciences; no. 70182) and overlaid with
sections using a Leica CM1850 microtome at ?16°C. The sections were
TABLE 1 Strains and plasmids used in this studya
Strains/plasmidsCharacteristics Source or reference
PA14 ?phz YFP
Clinical isolate UCBPP-PA14
PA14 with deletions of operons phzA1-G1 and phzA2-G2
PA14 with chromosomally integrated constitutive eYFP
PA14 ?phz with chromosomally integrated constitutive eYFP
DKN370; PA14 containing two copies of phzM
PA14 with a deletion of pelB-pelG
PA14 ?phz with a deletion of pelB-pelG
PA14 ?napA ?phz
PA14 ?narG ?phz
PA14 with a deletion of napA
PA14 with deletions of napA, phzA1-phzG1, and phzA2-phzG2
PA14 with a deletion of narG
PA14 with deletions of narG, phzA1-phzG1, and phzA2-phzG2
UQ950 E. coli DH5? ?(pir) host for cloning; F??(argF-lac)169 ?80dlacZ58(?M15) glnV44(AS)
rfbD1 gyrA96(Nalr) recA1 endA1 spoT1 thi-1 hsdR17 deoR ?pir?
Donor strain for conjugation: thrB1004 pro thi rpsL hsdS
lacZ ?M15RP4–1360 ?(araBAD)567 ?dapA1341::[erm pir(wt)]
InvSc1MATa/MAT? leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52 his3-?1/his3-?1 23; Invitrogen
Yeast-based allelic-exchange vector; sacBaCEN/ARSH URA3?Genr
Yeast-based expression vector 2?m; URA3?Genr
napA deletion fragments cloned into pMQ30
narG deletion fragments cloned into pMQ30
pMQ72 with napEFDABC operon insert
aTransposon mutants used in this study were confirmed by PCR or sequencing.
Redox Homeostasis in Structured Bacterial Communities
April 2013 Volume 195 Number 7jb.asm.org 1373
collected on Thermo Superfrost Plus slides (no. 6776214) and stored at
?80°C until imaging.
Thin sections were photographed using a Zeiss Axio Imager 2. All
images were obtained at ?10 magnification using a Zeiss EC-Plan Neo-
fluar objective with differential interference contrast (DIC) and fluores-
cence optics. The images were taken at an exposure time of 328 ms, false
colored, and processed in Photoshop CS4 (Adobe).
ized Clark-type oxygen sensor (Unisense; 10-?m tip diameter). The elec-
trode was connected to a picoampere amplifier multimeter (Unisense)
and polarized with ?0.8 V. The sensor was calibrated using a two-point
calibration system. The atmospheric oxygen reading was obtained by
placing the electrode in a calibration chamber (Unisense) that contained
well-aerated deionized water. Complete aeration was achieved by con-
stantly bubbling the water with air. The zero reading was obtained by
bubbling water in the calibration chamber with ultra-high-purity nitro-
gen gas (TechAir). All calibration readings and profile measurements
were obtained using SensorTrace pro 2.0 software (Unisense).
NADH/NAD?assay. Extraction and quantification of NADH and
NAD?were carried out according to the methods described by San et al.
(24) and Bernofsky and Swan (25). For cultures grown in LB, two 1-ml
centrifuged at 16,000 ? g for 1 min. Colonies grown on 1% tryptone and
sie blue dyes were scraped off the agar plate at the indicated time points
using sterile razor blades and resuspended in 1 ml of 1% tryptone. The
colonies were disrupted using a pellet disrupter. For each resuspended
colony, two 450-?l samples were placed into two separate microcentri-
fuge tubes. NADH and NAD?were then extracted from the liquid cul-
ture- or colony-derived samples, and relative or absolute quantification
was carried out using an enzyme-cycling assay, as described by Price-
Whelan et al. (26).
RESULTS AND DISCUSSION
In this study, we set out to characterize the relationship between
biofilm development. This work supports the hypothesis that
phenazine production and colony rugosity are adaptations that
facilitate survival by mitigating electron acceptor limitation. In
homogeneous liquid cultures of P. aeruginosa, phenazines affect
gene expression and oxidize the intracellular redox state (26–28).
dependent electron transfer between cells and an oxidizing elec-
trode supports survival (21). Given that cells in biofilms experi-
ence steep gradients in oxygen availability, leading to hypoxia or
morphological switch observed in phenazine-deficient colonies is
related to their limited ability to access oxidants.
To test this hypothesis, we first characterized the depth of ox-
ygen penetration within our colony biofilm system. P. aeruginosa
phenazine-null (?phz) biofilms characteristically increase their
produce more exopolysaccharides than wild-type colonies (7).
We therefore asked whether dissolved oxygen concentrations dif-
fered in the wild-type and ?phz biofilms when they were grown
and 40 ?g/ml Congo red). Measurements taken with a Clark ox-
ygen electrode (10-?m tip) revealed steep gradients, with oxygen
microelectrode measurements of P. aeruginosa biofilms grown in
effects of consumption and diffusion limitation (20). The extent
of oxygen depletion in the base remained constant over 4 days
kles, its slope was lower and decreased over time compared to the
base. We found that wrinkle thickness correlated with oxygen
abundance: thinner wrinkles were less oxygen depleted than
thicker wrinkles (Fig. 1D).
While respiratory versatility is a hallmark of some bacterial
species, P. aeruginosa is relatively limited in this regard. It can
grow by aerobic respiration and denitrification and poorly by ar-
ginine fermentation (29); therefore, the major energy-generating
metabolism contributing to growth of colonies on 1% tryptone is
aerobic respiration. Moreover, it is generally believed that in the
organic-rich environment of infections—such as the mucus that
collects on the lungs of individuals with cystic fibrosis—growth
and survival of Pseudomonas is not carbon/electron limited but
constrained by oxygen availability (30). Microelectrode measure-
ments showed oxygen depletion for both wild-type and ?phz col-
onies 60 ?m from the surface (Fig. 1B), although wild-type colo-
were comparable, expecting the cells to be confined to the region
within 60 ?m from the colony surface, where oxygen was avail-
TABLE 2 Primers used in this study
Primer Sequence (5= to 3=)
?napA 5= flank-1 GGAATTGTGAGCGGATAACAATTTCACA
?napA 5= flank-2
?napA 3= flank-1
?napA 3= flank-2
?narG 5= flank-1 GGAATTGTGAGCGGATAACAATTTCACA
?narG 5= flank-2
?narG 3= flank-1
?narG 3= flank-2
Dietrich et al.
jb.asm.orgJournal of Bacteriology
and the ?phz mutant that constitutively expressed a stable yellow
fluorescent protein (YFP). Colonies were fixed under oxic condi-
tions with paraformaldehyde and embedded in Tissue-Tek OCT,
frozen, and sectioned at a thickness of 10 ?m (Fig. 2A to F). Be-
were previously anoxic.
In sections taken from ?phz colonies, cells were found within
60 ?m from the surface, in the oxic zone (Fig. 2G). In sections
taken from wild-type colonies, however, cells were found up to
100 ?m from the surface (Fig. 2G). Some of these cells, therefore,
inhabited the 40-?m-thick anoxic zone. Reasoning that
phenazines enable this survival by acting as alternate electron ac-
ceptors in the absence of oxygen, we altered the ambient oxygen
concentration and predicted that the extent of the oxic zone
spheric oxygen levels). We grew colonies under hyperoxic and
hypoxic conditions (40% and 15% oxygen, respectively) and ob-
served that the depth at which cells could be detected in both
wild-type and ?phz colonies correlated with the concentration of
oxygen provided (Fig. 2A to F). Quantification of the cell layer
We next evaluated whether oxygen accessibility affects other
and wrinkle thickness of colonies grown with various concentra-
tions of oxygen. As oxygen concentrations increased, the colonies
FIG 2 Oxygen and phenazine availability modulates colony morphology. (A to F) Oxygen and phenazines affect cell layer thickness in P. aeruginosa colonies.
embedded in Tissue-Tek OCT. The images were taken using fluorescence (YFP) and DIC microscopy. The scale bar represents 200 ?m. (G) Depth at which the
YFP signal was detected in the sample. The error bars represent standard deviations for the means of measurements for 15 bases or wrinkles from 3 colonies. P
values were calculated using the one-tailed heteroscedastic Student’s t test comparing YFP signal depth in WT or ?phz colonies at different oxygen concentra-
tions. The least significant differences are shown. (H) Colony morphology. Representative images of ?phz colonies grown at various atmospheric oxygen
concentrations over 3 days. The scale bar represents 1 cm. (I) Colony wrinkle width (top) and surface coverage (bottom) for ?phz colonies grown at various
oxygen concentrations, with nitrogen comprising the atmospheric balance. The error bars represent the standard deviations of widths for 15 wrinkles from 3
colonies (top) or of measurements taken from 5 colonies (bottom).
Redox Homeostasis in Structured Bacterial Communities
April 2013 Volume 195 Number 7jb.asm.org 1375
I). To further probe the link between oxidant availability and col-
ony structure, we took advantage of P. aeruginosa’s ability to use
nitrate instead of oxygen for respiration and redox homeostasis.
Two P. aeruginosa nitrate reductase complexes, Nar and Nap,
might be expected to affect the intracellular redox state under
hypoxic conditions. While Nar is a cytoplasmic, membrane-asso-
ciated complex that contributes to the production of a proton
and is thought to balance the intracellular redox state without
directly contributing to the generation of a transmembrane elec-
trochemical gradient (33, 34). Both Nap and Nar catalyze the re-
duction of nitrate to nitrite. Three additional enzyme complexes,
Nos (nitrous oxide reductase), allow P. aeruginosa to perform
denitrification, the full reduction of nitrate to nitrogen gas (N2).
amended with 40 mM potassium nitrate and found that these
conditions rendered the colony smooth (Fig. 3A). To determine
whether this was due to nitrate reduction, we generated mutants
lacking the nitrate reductase subunits NarG and NapA. The
?narG mutant formed a smooth colony when grown on nitrate,
suggesting that nitrate respiration is not required for the nitrate-
tant wrinkled when grown on medium amended with nitrate
(Fig. 3A) but reverted to smooth when complemented by the nap
operon on a plasmid (see Fig. S2 in the supplemental material).
We then tested whether the downstream enzymes in the denitri-
ness. Mutants deficient in Nir and Nor also formed wrinkled col-
bar represents 1 cm. (B) Colony morphology of denitrification mutants. Specific steps in the canonical denitrification pathway catalyzed by Nap, Nir, and Nor
represents 1 cm. (C) PCA rescues the ?napA phenotype on nitrate; addition of exogenous PCA promotes smoothness in a ?napA ?phz mutant. The images are
representative of 3 independent experiments. The scale bar represents 1 cm.
FIG 4 (A) NADH/NAD?ratios for liquid cultures grown for 16 h. For 40 mM
blue pigmentation (pyocyanin production) was apparent in the wild-type cul-
tures. The error bars represent the standard deviations of triplicate cultures. (B)
(Top) NADH/NAD?for wild-type and ?phz colonies. The error bars represent
The scale bar represents 0.5 cm. The images are representative of 3 independent
Dietrich et al.
jb.asm.orgJournal of Bacteriology
onies on 40 mM nitrate, while the mutant lacking functional Nos
remained smooth, suggesting that reduction to nitrous oxide is
required for NapA-dependent nitrate reduction (Fig. 3B). Fur-
thermore, these results suggest that P. aeruginosa community
structure is determined, at least in part, by the intracellular redox
If both phenazine reduction and nitrate reduction contribute
to oxidizing the intracellular redox state, one would predict that
ony smoothness. Nitrate addition to a ?phz mutant decreased
wrinkling (Fig. 3A), and phenazine-1-carboxylic acid (PCA) ad-
also had this effect (Fig. 3C).
To further test the hypothesis that community structure and
the intracellular redox state are linked, we set out to measure and
manipulate the NADH-to-NAD?ratio in colony biofilms under
uid cultures (26), we first asked whether nitrate-dependent redox
balancing required napA. We extracted NADH and NAD?from
planktonically grown cells and measured their levels using an en-
zyme-based cycling assay (24, 25). We found that the ?napA mu-
tant showed a partial but significant defect in nitrate-dependent
type P. aeruginosa (Fig. 4A). We next adapted the NADH/NAD?
extraction and quantification protocol for use with colony sam-
ples. This method revealed that the NADH/NAD?ratio of phen-
azine-null colonies reached a maximum that coincided with the
induction of wrinkling, while the NADH/NAD?ratio of wild-
type colonies remained relatively consistent throughout the time
course (Fig. 4B). Absolute quantitation of the NADH and NAD?
total NAD(H) pools of cells in wild-type and ?phz colonies were
potential reaches a threshold value, indicating that increased col-
promotes rebalancing of the intracellular redox state. To test this
we observed a correlation between wrinkle formation and colony
staining with Congo red, suggesting that production of the PEL
obtained ?pel mutants lacking genes required for PEL biosynthe-
sis to test in our colony biofilm assay (35). As expected, colony
both wild-type and ?phz backgrounds (Fig. 5A). We then mea-
sured NADH/NAD?ratios during colony maturation and found
that the transient increased ratio that coincided with the onset of
FIG 5 (A) PEL production is required for wrinkle formation. pelB-pelG were deleted in the wild-type and ?phz backgrounds. Colonies were grown for 5 days on 1%
comparing ?phz and ?phz ?pel colonies at 42 and 48 h. (C) NADH/NAD?ratios in liquid culture (early stationary phase) for wild-type, ?phz, ?pel, and ?phz ?pel
Redox Homeostasis in Structured Bacterial Communities
April 2013 Volume 195 Number 7 jb.asm.org 1377
wrinkling in the ?phz mutant persisted in the ?phz ?pel mutant
(Fig. 5B). In contrast, the pel deletions had no effect on the
supports the hypothesis that wrinkling is a strategy for balancing
the intracellular redox state of cells within a community. These
results suggest that colony morphological development is an ac-
tive process in which a critical redox state is sensed, leading to a
biological response. We are in the process of identifying the cir-
cuitry responsible for this phenomenon and the extent to which
wrinkling is an emergent property. We note that the specific time
when the NADH/NAD?ratio peaks can vary from experiment to
experiment as a function of slight differences in plate thickness,
density of the initial inoculum, etc. However, occurrence of the
peak just prior to colony wrinkling is highly reproducible.
All cells catalyze a repertoire of catabolic and anabolic reac-
tions that must be balanced so that the cytoplasm remains a hos-
redox reactions have been identified that appear to serve the sole
purpose of modulating the intracellular redox state, i.e., they do
not contribute directly to energy generation or the production of
biomass. We have shown that P. aeruginosa can use endogenous
phenazines and/or exogenous nitrate to balance the intracellular
unable to produce phenazines form structurally more complex
communities with increased surface area led us to propose that
this morphogenetic switch is a response to a reduced cytoplasm.
ity—and thereby redox balance—in a bacterial system (Fig. 6).
Furthermore, inhibiting this response disrupts redox balancing
(Fig. 5B). Whether rugosity has a similar effect in other microbial
species remains to be investigated. It would not be surprising if
bacteria with different metabolic properties (e.g., with or without
the ability to produce electron shuttles) have different mecha-
nisms for growing and surviving in multicellular communities.
Intriguingly, a recent study of patterning in Bacillus subtilis bio-
films (36) suggested that localized cell death promotes wrinkle
but to our knowledge, experiments have not yet been performed
that enable a direct comparison. Understanding how active pro-
cesses and passive physical effects interweave to achieve multicel-
lular patterning in Pseudomonas is our long-term goal; it will be
interesting to learn whether the mechanisms that underpin these
patterns are generalizable. As has been well articulated by others
(37), the complex interplay between physical, chemical, and bio-
Dietrich et al.
jb.asm.orgJournal of Bacteriology
for future research.
In conclusion, as Friedheim recognized nearly a century ago
(15), cellular aggregation leads to gradient formation due to lim-
ited diffusion and consumption of substrates by individual cells
eukaryotic tissues, cells in multicellular environments likely em-
ploy differing strategies to ensure substrate acquisition and sur-
vival, depending on the specific microenvironment they inhabit.
reports have discussed the fact that oxygen defines metabolic
zones in biofilms (19, 20, 38). Here, for the first time, we have
development. Mechanisms that aid in redox homeostasis at the
cellular level have been characterized in diverse organisms. In
metazoans, redox-balancing mechanisms that function at the
multicellular level are also well known; for example, the develop-
ment of the vascular system prevents oxygen starvation of the
growing embryo (39). In multicellular aerobes, cells must cope
with limited oxygen availability that leads to the formation of
aerobic, microaerobic, and anaerobic zones. During processes
such as tumor angiogenesis, relative oxygen concentrations act as
cues that determine adaptive morphological features, facilitating
oxygen delivery to cells within the macroscopic structure (40).
Our findings suggest that, like metazoans, bacteria can also re-
spond to electron acceptor limitation and balance intracellular
redox levels through morphological changes at the community
level. Morphological adaptation to redox imbalance thus appears
to be a conserved biological strategy.
We thank W. Ziebis, P. Girguis, G. Squyres, and D. Chapman for
This research was supported by funding from the Howard Hughes
Medical Institute (HHMI) (L.E.P.D., A.P.-W., R.C.H., and D.K.N.), a
NSF (H.S.), and a startup fund from Columbia University to
L.E.P.D. D.K.N. is an HHMI Investigator.
1. Grosberg RK, Strathmann RR. 2007. The evolution of multicellularity: a
minor major transition? Annu. Rev. Ecol. Evol. Systematics 38:621–654.
2. Orphan VJ, House CH, Hinrichs KU, McKeegan KD, DeLong EF. 2002.
Multiple archaeal groups mediate methane oxidation in anoxic cold seep
sediments. Proc. Natl. Acad. Sci. U. S. A. 99:7663–7668.
3. Lopez D, Vlamakis H, Kolter R. 2010. Biofilms. Cold Spring Harb.
Perspect. Biol. 2:a000398. doi:10.1101/cshperspect.a000398.
4. Stewart PS, Franklin MJ. 2008. Physiological heterogeneity in biofilms.
Nat. Rev. Microbiol. 6:199–210.
5. Booth SC, Workentine ML, Wen J, Shaykhutdinov R, Vogel HJ, Ceri H,
Turner RJ, Weljie AM. 2011. Differences in metabolism between the
biofilm and planktonic response to metal stress. J. Proteome Res. 10:
nas aeruginosa infection. J. Cyst. Fibros. 4(Suppl. 2):49–54.
7. Dietrich LE, Teal TK, Price-Whelan A, Newman DK. 2008. Redox-
active antibiotics control gene expression and community behavior in
divergent bacteria. Science 321:1203–1206.
8. Ramos I, Dietrich LE, Price-Whelan A, Newman DK. 2010. Phenazines
affect biofilm formation by Pseudomonas aeruginosa in similar ways at
various scales. Res. Microbiol. 161:187–191.
9. Price-Whelan A, Dietrich LE, Newman DK. 2006. Rethinking ‘second-
10. Mavrodi DV, Peever TL, Mavrodi OV, Parejko JA, Raaijmakers JM,
Lemanceau P, Mazurier S, Heide L, Blankenfeldt W, Weller DM,
Thomashow LS. 2010. Diversity and evolution of the phenazine biosyn-
thesis pathway. Appl. Environ. Microbiol. 76:866–879.
11. Wang Y, Newman DK. 2008. Redox reactions of phenazine antibiotics
with ferric (hydr)oxides and molecular oxygen. Environ. Sci. Technol.
13. Harrop GA, Barron ES. 1928. Studies on blood cell metabolism. I. The
effect of methylene blue and other dyes upon the oxygen consumption of
mammalian and avian erythrocytes. J. Exp. Med. 48:207–223.
14. Barron ES, Hoffman LA. 1930. The catalytic effect of dyes on the oxygen
consumption of living cells. J. Gen. Physiol. 13:483–494.
15. Friedheim EA. 1934. The effect of pyocyanine on the respiration of some
normal tissues and tumours. Biochem. J. 28:173–179.
16. Barron ES. 1930. The catalytic effect of methylene blue on the oxygen
consumption of tumors and normal tissues. J. Exp. Med. 52:447–456.
17. Kerr JR. 2000. Phenazine pigments: antibiotics and virulence factors.
Infect. Dis. Rev. 2:184–194.
18. Hernandez ME, Newman DK. 2001. Extracellular electron transfer. Cell.
Mol. Life Sci. 58:1562–1571.
19. Peters AC, Wimpenny JW, Coombs JP. 1987. Oxygen profiles in, and in
erichia coli. J. Gen. Microbiol. 133:1257–1263.
20. Xu KD, Stewart PS, Xia F, Huang CT, McFeters GA. 1998. Spatial
physiological heterogeneity in Pseudomonas aeruginosa biofilm is deter-
mined by oxygen availability. Appl. Environ. Microbiol. 64:4035–4039.
21. Wang Y, Kern SE, Newman DK. 2010. Endogenous phenazine antibiot-
ics promote anaerobic survival of Pseudomonas aeruginosa via extracellu-
lar electron transfer. J. Bacteriol. 192:365–369.
22. Bertani G. 2004. Lysogeny at mid-twentieth century: P1, P2, and other
experimental systems. J. Bacteriol. 186:595–600.
23. Shanks RM, Caiazza NC, Hinsa SM, Toutain CM, O’Toole GA. 2006.
Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes
24. San KY, Bennett GN, Berrios-Rivera SJ, Vadali RV, Yang YT, Horton E,
Rudolph FB, Sariyar B, Blackwood K. 2002. Metabolic engineering
through cofactor manipulation and its effects on metabolic flux redistri-
bution in Escherichia coli. Metab. Eng. 4:182–192.
25. Bernofsky C, Swan M. 1973. An improved cycling assay for nicotinamide
adenine dinucleotide. Anal. Biochem. 53:452–458.
domonas aeruginosa PA14. J. Bacteriol. 189:6372–6381.
27. Dietrich LE, Price-Whelan A, Petersen A, Whiteley M, Newman DK.
2006. The phenazine pyocyanin is a terminal signalling factor in the quo-
rum sensing network of Pseudomonas aeruginosa. Mol. Microbiol. 61:
28. Sullivan NL, Tzeranis DS, Wang Y, So PT, Newman D. 2011. Quanti-
fying the dynamics of bacterial secondary metabolites by spectral mul-
tiphoton microscopy. ACS Chem. Biol. 6:893–899.
29. Vander Wauven C, Pierard A, Kley-Raymann M, Haas D. 1984. Pseu-
domonas aeruginosa mutants affected in anaerobic growth on arginine:
J. Bacteriol. 160:928–934.
30. Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC,
Birrer P, Bellon G, Berger J, Weiss T, Botzenhart K, Yankaskas JR,
Randell S, Boucher RC, Doring G. 2002. Effects of reduced mucus
oxygen concentration in airway Pseudomonas infections of cystic fibrosis
patients. J. Clin. Invest. 109:317–325.
31. Heim R, Prasher DC, Tsien RY. 1994. Wavelength mutations and post-
translational autoxidation of green fluorescent protein. Proc. Natl. Acad.
Sci. U. S. A. 91:12501–12504.
32. Zhang C, Xing X-H, Lou K. 2005. Rapid detection of a GFP-marked
Enterobacter aerogenes under anaerobic conditions by aerobic fluores-
cence recovery. FEMS Microbiol. Lett. 249:211–218.
33. Williams HD, Zlosnik JE, Ryall B. 2007. Oxygen, cyanide and energy
generation in the cystic fibrosis pathogen Pseudomonas aeruginosa. Adv.
Microb. Physiol. 52:1–71.
34. Richardson DJ, Berks BC, Russell DA, Spiro S, Taylor CJ. 2001. Func-
Cell. Mol. Life Sci. 58:165–178.
Redox Homeostasis in Structured Bacterial Communities
April 2013 Volume 195 Number 7 jb.asm.org 1379
35. Friedman L, Kolter R. 2004. Genes involved in matrix formation in Download full-text
Pseudomonas aeruginosa PA14 biofilms. Mol. Microbiol. 51:675–690.
36. Asally M, Kittisopikul M, Rue P, Du Y, Hu Z, Cagatay T, Robinson AB,
Lu H, Garcia-Ojalvo J, Suel GM. 2012. Localized cell death focuses
U. S. A. 109:18891–18896.
37. Klapper I, Dockery J. 2010. Mathematical description of microbial bio-
films. Siam Rev. 52:221–265.
38. Xavier JB, Foster KR. 2007. Cooperation and conflict in microbial bio-
films. Proc. Natl. Acad. Sci. U. S. A. 104:876–881.
39. Simon MC, Keith B. 2008. The role of oxygen availability in embry-
onic development and stem cell function. Nat. Rev. Mol. Cell Biol.
of oxygen sensing in development, normal function, and disease. Genes
41. Lambertsen L, Sternberg C, Molin S. 2004. Mini-Tn7 transposons for
42. Rahme LG, Stevens EJ, Wolfort SF, Shao J, Tompkins RG, Ausubel FM.
1995. Common virulence factors for bacterial pathogenicity in plants and
animals. Science 268:1899–1902.
43. Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E, Wu G,
Villanueva J, Wei T, Ausubel FM. 2006. An ordered, nonredundant
library of Pseudomonas aeruginosa strain PA14 transposon insertion mu-
tants. Proc. Natl. Acad. Sci. U. S. A. 103:2833–2838.
Dietrich et al.
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