A bacterial symbiont is converted from an inedible
producer of beneficial molecules into food by a single
mutation in the gacA gene
Pierre Stallfortha, Debra A. Brockb, Alexandra M. Cantleya, Xiangjun Tianb, David C. Quellerb, Joan E. Strassmannb,
and Jon Clardya,1
aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; andbDepartment of Biology, Washington
University in St. Louis, St. Louis, MO 63130
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved July 3, 2013 (received for review May 1, 2013)
Stable multipartite mutualistic associations require that all part-
ners benefit. We show that a single mutational step is sufficient to
turn a symbiotic bacterium from an inedible but host-beneficial
secondary metabolite producer into a host food source. The bac-
teria’s host is a “farmer” clone of the social amoeba Dictyostelium
discoideumthat carries and disperses bacteria during its spore stage.
Associated with the farmer are two strains of Pseudomonas fluores-
cens, only one of which serves as a food source. The other strain
agent, and a chromene that potently enhances the farmer’s spore
production and depresses a nonfarmer’s spore production. Genome
sequence and phylogenetic analyses identify a derived point muta-
tion in the food strain that generates a premature stop codon in
a global activator (gacA), encoding the response regulator of a two-
component regulatory system. Generation of a knockout mutant of
this regulatory gene in the nonfood bacterial strain altered its sec-
ondary metabolite profile to match that of the food strain, and also,
independently,converteditintoa foodsource.These resultssuggest
tective role converted it to a “domesticated” food source.
symbiosis|GacA–GacS two-component system|differential metabolomics
cinating set of structurally diverse molecules that defend the
host, initiate host developmental changes, and carry out other
important functions have been shaped by their evolutionary
history (1–10). Recently, Brock et al. (11) described an associa-
tion between the social amoeba Dictyostelium discoideum and a
variety of Gram-negative bacteria, some of which it carries to
initiate new food populations.
D. discoideum is a popular model for studying multicellularity,
chemical signaling, general eukaryotic cellular mechanisms, and
social phenomena (12–15). The protist is typically found in soil,
where it preys on bacteria; in nutrient-rich environments it lives
as single-celled organisms that reproduce by binary fission. Upon
starvation, cAMP-mediated aggregation occurs, leading to the
formation of a multicellular pseudoplasmodium containing up to
105individual cells. Eventually, the aggregate develops into a
fruiting body in which some 20% of the cells differentiate into
a dead stalk that supports a spherical structure known as the
sorus; the latter contains 80% of the cells that turn into spores.
Previous work by Brock et al. (11) showed that about one-third
of wild-collected clones of D. discoideum engage in stable asso-
ciations with bacteria throughout the sporulation and dispersal
process. These clones are called “primitive farmers” because
they carry, seed, and prudently harvest their bacterial food.
Schultz and Brady (16) characterized agriculture as a special-
ized form of symbiosis known in only four animal groups: humans,
bark beetles, termites, and ants. The fungus-farming ants, for
example, collect material above ground and carry it to their
underground fungal gardens. The ants and the fungi are
mall molecules regulate mutually beneficial associations be-
tween bacterial symbionts and their eukaryotic hosts. A fas-
obligate mutualists; the fungus requires the ants, and the ants
require the fungus (8). In addition, the ants have specialized
anatomical features that contain bacteria (actinomycetes be-
longing to the genus Pseudonocardia) that produce small-molecule
chemical defenses that protect the fungal crop from specialized
fungal pathogens (5). This multipartite symbiosis has existed for
∼50 million years and evolved into more than 230 species. The
bark beetle system also involves an obligate mutualism between
a fungal food source and a beetle host, along with Streptomyces
spp. bacteria that provide chemical defenses, and this system has
∼200 genera and ∼6,000 species.
The initial model of primitive farming in D. discoideum con-
tained a puzzling feature: only about half of the bacteria carried
by the farmers served as food sources. Though it made sense that
the farmer clones would carry bacterial food sources, it was not
clear why they should also carry bacteria that they cannot eat. It
seemed likely that we could resolve this question by exploring the
differences between bacterial strains with similar genetic back-
grounds but different functional roles.
In our study, we focused on a single tripartite association be-
tween one farmer D. discoideum clone and its two carried strains
of the Gram-negative gammaproteobacterium Pseudomonas flu-
orescens. One of the P. fluorescens strains served as a food source
for the farmer and the other one did not. In this study, we iden-
tified the role of the nonfood source in the symbiosis, as well as the
genetic difference between food and nonfood P. fluorescens. Fi-
nally, a phylogenetic analysis allowed us to retrace the evolu-
tionary history of the two bacterial strains.
Results and Discussion
Metabolomic and Structural Investigation of Symbiotic P. fluorescens
Strains. To determine why D. discoideum would carry bacteria
that did not serve as a food source, we investigated the pro-
duction of secondary metabolites by the two symbiotic P. fluo-
rescens strains associated with the host-farmer D. discoideum
QS161 (from now on referred to as the “farmer”). Both sym-
bionts had identical 16S rRNA gene sequences; however, one
strain, P. fluorescens PfB-QS161 (from now on referred to as
“PfB”), served as a food source, and the other one, P. fluorescens
PfA-QS161 (from now referred to as “PfA”), did not (SI Ap-
pendix, Fig. S1). We used a differential metabolomics approach
Author contributions: P.S., D.A.B., A.M.C., X.T., D.C.Q., J.E.S., and J.C. designed research;
P.S., D.A.B., A.M.C., and X.T. performed research; P.S., D.A.B., A.M.C., X.T., D.C.Q., J.E.S., and
J.C. analyzed data; and P.S., D.A.B., A.M.C., X.T., D.C.Q., J.E.S., and J.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
See Commentary on page 14512.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 3, 2013
| vol. 110
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to identify molecules with potential functional roles in the sym-
biosis (Fig. 1).
We grew the food source strain PfB and the nonfood source
strain PfA in liquid media for 24 h. Initial LC-MS analysis of the
ethyl acetate extract of the PfA culture revealed two major peaks
that correspond to two different compounds: a previously un-
reported compound and the known antifungal pyrrolnitrin (Fig.
1) (17). The extracts of the PfB culture revealed one major dif-
fusible small molecule—namely, the iron chelator pyochelin (Fig. 1)
(18). The latter contains three stereogenic centers of which one
(C2′′) can easily epimerize to yield a mixture of diastereoisomers
known as pyochelin I and II. Furthermore, pseudomonads are
known to produce two enantiomeric (mirror image) forms of
pyochelin: (4′S, 4′′S) and (4′R, 4′′R), depending on whether they
contain a cysteine epimerase domain in their biosynthetic gene
cluster (19). Whereas fluorescent pseudomonads biosynthesize
(4′S, 4′′S) pyochelin, P. aeruginosa, biosynthesizes (4′R, 4′′R)
pyochelin (19). A measurement of the optical rotation revealed
that PfB produces the enantiomer characteristic of fluorescent
The unknown compound found in the PfA extract was pro-
duced in liquid culture at a concentration of ∼0.3 μg/mL (1.3
μM) after 24 h of cultivation at 30 °C; it was isolated as a pale
yellow oil with the empirical formula C14H18O3as determined by
high-resolution electrospray ionization-TOF mass spectrometry
combined with both1H and13C NMR spectroscopy (SI Appen-
dix, Table S1 and Figs. S2 and S3; Fig. 1). The1H NMR spec-
trum in deuterated methanol displayed three downfield protons
at 6.33, 5.86, and 5.78 ppm, a double doublet at 4.51 ppm, and 12
aliphatic protons between 0 and 3 ppm. The
gradient heteronuclear single-quantum coherence (gHSQC) spec-
tra showed five aromatic, two olefinic, one oxygen-bearing ali-
phatic, and five aliphatic carbon signals. A combination of1H-1H
correlation spectroscopy (COSY), gHSQC, and heteronuclear
multiple-bond correlation (HMBC) spectra allowed us to identify
13C NMR and
the molecule as the previously undescribed 3-ethyl-2-propyl-2H-
chromene-5,7-diol (hereafter referred to as “chromene”; Fig. 1).
We confirmed the identity of both pyrrolnitrin and enantio-
pyochelin by a combination of high-resolution mass spectrometry
published data (SI Appendix, Fig. S5 and Table S2). We then
investigated the role of the main secondary metabolites produced
by the nonfood strain PfA in the symbiotic association with the
1H NMR spectroscopy data, which were compared with
Differential Effect of Pyrrolnitrin on Farmer and Nonfarmer
D. discoideum. In many symbiotic associations between bacteria
and eukaryotes, the former produce secondary metabolites that
protect their host from pathogens or display other beneficial
effects. Pyrrolnitrin, which is produced by PfA, the nonfood
source symbiont of D. discoideum, has potent antifungal activity
against a variety of soil-borne fungi (20); it is also known to play
a crucial role in other symbiotic associations between the pro-
ducer P. fluorescens strain and plants. The decreased suscepti-
bility of an infected host to soil-borne pathogens can be used as
a form of biocontrol, and fluorescent pseudomonads play an
important role as biocontrol agents (21–24). It is possible that
Dictyostelia carrying pyrrolnitrin-producing strains like PfA could
be similarly protected.
The discovery of robust pyrrolnitrin production by PfA (0.4 μg/
mL = 1.6 μM in a 24-h liquid culture) was in some ways also
surprising because this metabolite is potentially toxic to many
eukaryotes, including D. discoideum. In one recent study on
predator–prey chemical warfare involving P. fluorescens as the
prey and another amoeba (Acanthamoeba castellanii) as the
predator, pyrrolnitrin was toxic, and supernatants of amoeba
cultures strongly induced the production of the molecule (25).
Farmer resistance and nonfarmer sensitivity would indicate that
the farmers adapted to harboring a potentially lethal symbiont.
+ Spore production
- Spore production
S ST TOP OP
the farmer, the former does not. Both strains produce different secondary metabolites: PfA produces a chromene and pyrrolnitrin, whereas PfB produces the
putative siderophore (iron chelator) pyochelin.
Two strains are carried by the farmer D. discoideum QS161: P. fluorescens PfA (Upper) and PfB (Lower). Though the latter serves as a food source for
Stallforth et al. PNAS
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To test whether the farmer has evolved the ability to resist the
toxicity of its bacterial symbiont as a consequence of continuous
exposure to its metabolites, we subjected the farmer D. discoideum
strain to a range of concentrations of pyrrolnitrin. As a control, we
investigated the effect of this compound on a nonhost, non-
farmer clone, D. discoideum QS160 (hereafter referred to as the
“nonfarmer”), which was isolated in the vicinity of the farmer
and was thus likely to have occasionally encountered the farmer
D. discoideum strain as well as its symbiotic bacteria (PfA and
PfB) and their secreted molecules. In D. discoideum, the number
of spores produced from a given number of amoebas is a good
indicator of the success of the social process, so we used this
measure to evaluate the effect of pyrrolnitrin on both D. discoideum
strains. To determine variation of spore production as a function
of the concentration of pyrrolnitrin, we put vegetative amoebas
in their exponential growth phase on a nitrocellulose filter paper
soaked with starvation buffer containing different concentrations
of pyrrolnitrin. Fruiting bodies form within 24 h; at this time
point we counted the number of spores contained within the
The farmer strain and the nonfarmer strain performed differ-
ently in the presence of pyrrolnitrin (Fig. 2). Though the non-
farmer suffered from the toxic effect of pyrrolnitrin, the farmer did
not, and in fact did better than controls. We interpret these results
as an adaptation of the farmer to pyrrolnitrin’s toxic effects.
Differential Effect of Chromene on Farmer and Nonfarmer D. discoideum.
The presence of pyrrolnitrin in PfA suggested that chromene
might have similar or related biocontrol activities. In a 24-h
liquid culture, chromene was found to be present in comparable
concentration to pyrrolnitrin (chromene: 0.3 μg/mL = 1.3 μM;
pyrrolnitrin: 0.4 μg/mL = 1.6 μM).
Initial bioassays, however, revealed no activity against the
model fungus Saccharomyces cerevisiae or the gammaproteo-
bacterium Escherichia coli, and only modest activity against Ba-
cillus subtilis (IC50= 82 μg/mL = 35 μM). Because we found no
obvious indirect effects (inhibition of D. discoideum’s microbial
competitors) for chromene, we investigated possible direct
effects on the farmer D. discoideum.
Treatment with chromene increased spore production in the
farmer strain (Fig. 3), whereas the same treatment resulted in
a decrease in spore production by the nonfarmer D. discoideum
strain. The onset of visible differences in spore production oc-
curred at concentrations on the order of 1 ng/mL (∼4 nM). This
concentration of chromene is likely to be ecologically relevant
because it is ∼300-fold lower than the concentration of chro-
mene in a 24-h culture of PfA.
Having determined the molecular basis for farmer-beneficial
effects of the nonfood symbiont PfA, we investigated the genetic
difference between this bacterium and the food source PfB.
A Single Mutation Determines both Chemical Profile Changes and
Edibility, but They Are Distinct Pleiotropic Effects. Because Pseu-
domonas fluorescens strains PfA and PfB both have identical 16S
rRNA gene sequences, it seemed plausible that they shared
a recent common ancestor. If so, only a few alterations in their
genomes might have resulted in their observed phenotypic dif-
ferences. Both PfA and PfB were thus characterized by Next
Generation Illumina sequencing, and the resulting reads were
assembled using the genome of Pf-5, which was sequenced in
2005, as a scaffold (26).
Because PfA and PfB had different secondary metabolite
profiles, it seemed plausible to expect differences in biosynthetic
genes of the two strains. We focused on the known biosynthetic
gene clusters for the metabolites pyrrolnitrin and enantio-pyo-
chelin. Though only PfA produces pyrrolnitrin, and only PfB
produces enantio-pyochelin, both PfA and PfB had identical
coding sequences for the biosynthetic gene cluster for pyrrolni-
trin and enantio-pyochelin. This finding indicates that the dif-
ference between the small molecules produced by the two strains
results from differences in regulation rather than biosynthetic
potential. As a result, we searched for regulatory genes that,
when mutated, could lead to the observed phenotypes. The
search focused on two-component systems as the most likely
regulators of gene expression in bacteria.
A canonical two-component system is composed of a histidine
sensor kinase and a cognate response regulator. Upon stimula-
tion, the sensor kinase undergoes autophosphorylation to yield
a phosphorylated histidine, which in turn activates the response
regulator by phosphotransfer to an aspartyl residue (27, 28). The
response regulator then mediates the output, typically a change
in gene expression (29).
We found a point mutation in PfB generating a premature
stop codon that resulted in a truncated gacA gene product (Fig.
4). A cytosine-to-thymine mutation was responsible for the
conversion of wild-type gacA glutamine-164 to a stop codon in
PfB’s gacA gene.
The GacA–GacS system is a well-known global regulator in
many pseudomonads. In the opportunistic pathogen P. aeruginosa,
for instance, the GacS–GacA two-component system regulates the
expression of acute and chronic virulence determinants (30). In
%-Change in spore production
relative to control
Pyrrolnitrin concentration (ng/mL)
* * * * * *
* * * *
and QS160, respectively. Each data point shows mean ± SD at the specified
concentration (three biological replicates; P < 0.05 two-tailed test paired
t test of three experimental vs. three control values for each point, paired by
experimental block, df = 2; SI Appendix, Table S3). For purposes of visuali-
zation, we fit the graph with a second-order smoothing polynomial with
four neighbors on each side using GraphPad Prism 6 (www.graphpad.com)
Effect of pyrrolnitrin on farmer and nonfarmer D. discoideum QS161
Chromene concentration (ng/mL)
%-Change in spore production
relative to control
Farmer QS 161
* * * * *
* * * *
farmer D. discoideum QS161 vs. nonfarmer clone QS160. Each data point
shows mean ± SD at the specified concentration (four biological replicates;
P < 0.05 two-tailed test paired t test of four experimental vs. four control
values for each point, paired by experimental block, df = 2; SI Appendix,
Table S3). For purposes of visualization, we fit the graph with a sigmoidal
curve using GraphPad Prism 6 (www.graphpad.com) software.
Effect of chromene produced by PfA on the spore production of
| www.pnas.org/cgi/doi/10.1073/pnas.1308199110Stallforth et al.
P. fluorescens, the GacA–GacS system is a global regulator for an-
tibiotic production (31, 32). Upon binding to an as-yet-unidentified
external signal, this regulatory system upregulates the production
of pyrrolnitrin, and downregulates the production of siderophores
such as pyochelin. Spontaneous mutations disabling the GacA–
GacS system have been reported in P. fluorescens that were mainly
characterized by a change in colony morphology (33), not unlike
the differences in morphology between the PfA and PfB strains in
this study. Furthermore, such mutations in pseudomonads are
known to occur in the rhizosphere of plants where they display
a role in competitive root colonization (34). The point mutation
observed in PfB introduces a premature stop codon upstream of
the helix-turn-helix motif required for DNA binding, and inability
to bind DNA prevents the GacS response regulator from acti-
vating transcription (35) (Fig. 4).
To verify that a mutation in this two-component system was
sufficient to explain the chemical and edibility differences be-
tween PfA and PfB, we deleted the entire gacA gene in PfA. A
clean deletion was obtained by allelic replacement (36). The
gacA knockout of PfA displayed the PfB phenotype: it could now
serve as a food source for the farmer D. discoideum (SI Appendix,
Fig. S1) and the secondary metabolite profile was the same as for
PfB (Fig. 5). However, a complementation of PfB with PfA su-
pernatant (containing pyrrolnitrin and chromene) did not turn
PfB into a nonfood source, so it seems unlikely that these two
secreted molecules are responsible for the inedibility of PfA (SI
Appendix, Fig. S10). Instead, the edibility difference between the
strains must result from some other, unknown downstream effect
of gacA, which is not surprising given that its inactivation affects
expression of 10% of the genes in the genome (32).
A single mutation transforms a beneficial but inedible P. flu-
orescens strain into a food source for D. discoideum. The nonfood
source produces secondary metabolites that provide chemical
defenses (pyrrolnitrin) and increase spore production in the
farmer (chromene and pyrrolnitrin). This mutation in the gacA
gene generates independent but closely related symbiotic strains
that perform strikingly different roles in the symbiosis.
Though the genetic difference between the two bacterial strains
was determined, a detailed phylogenetic analysis is required to
understand their evolutionary history.
The Evolution of Edibility. It seems likely that the functional gacA
gene is ancestral because there are many more possible loss-of-
function mutations than gain-of-function mutations. In support
of this hypothesis, we examined the gacA sequences from the 11
other P. fluorescens strains with sequenced genomes and also the
two additional P. fluorescens strains we have isolated from
other D. discoideum farmer clones (Pf-QS68, Pf-QS152). All gacA
sequences appeared functional; none had the stop codon at po-
sition 164 or any other evident disabling stop codon or frameshift-
causing insertion or deletion.
Thus, the stop mutation that causes edibility is clearly derived.
The similarity of PfA and PfB suggests they are very closely re-
lated, yet both of them display a significant number of SNPs (136
in 5,561 homologous genes) showing they diverged well before
we isolated them in the laboratory. We then estimated the
maximum-likelihood phylogeny using DNA sequences of 20 con-
served genes for all 15 strains, using Pseudomonas syringae as an
out-group. The result (Fig. 6) shows very clear support (100%
bootstrap) for a clade that includes the previously sequenced
Pf-5 along with the four strains collected from D. discoideum.
a truncated protein. The latter is characterized by the loss of the highly conserved helix turn helix motif, which is required for DNA binding.
Amino acid sequence alignment of the gacA gene product of both PfA and PfB shows that a premature stop codon in the gacA gene of PfB leads to
5 10 15 20 min
ΔgacA (Middle), and PfA (Bottom) cultures. Though PfB and PfA ΔgacA
display virtually identical traces, PfA is distinctly different: PfA produces
chromene and pyrrolnitrin, whereas PfB and PfA ΔgacA both produce the
enantiomers of pyochelin I and II.
HPLC (254 nm) trace of an ethyl acetate extract of PfB (Top), PfA
Stallforth et al.PNAS
| September 3, 2013
| vol. 110
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There is some uncertainty (50% bootstrap) about the relative
positions of the outermost clones in the clade (Pf-5 and Pf-QS68).
This best tree is consistent with either two gains of the trait of
being carried by D. discoideum or with a single gain plus a loss in
Pf5, and because of uncertainty at the key node, we also cannot
rule out a less-supported tree with Pf5 basal to the other four,
with a single gain.
The tree also strongly supports PfA and PfB being the closest
relatives whose sister group is another farmer-carried clone, Pf-
QS152. Although the result could change with additional clone
sampling, the most parsimonious conclusion is the edibility of
PfB evolved in a strain already being carried by D. discoideum.
This analysis suggests the unusual result that an organism evolved
to be eaten by another, which makes sense only through kin-
selected benefits to clonemates, more of whom will be carried
to new locations by the well-fed farmer D. discoideum clone.
Conclusions and Perspective. The original description of primitive
farming by the social amoeba D. discoideum distinguished two
types of amoebas: farmers, which had fruiting bodies containing
several types of bacteria, and nonfarmers, which were bacteria-
free (11). Roughly half of the farmer-associated bacteria could
serve as a food source, and farmers transported to bacteria-poor
environments were able to sow food bacteria and eventually
consume this bacterial food. Using a differential metabolomics
analysis coupled with metabolite isolation and characterization,
we showed that a nonfood source bacterial symbiont (PfA) pro-
duces secondary metabolites that are beneficial for the farmer.
One of the metabolites, chromene, functions directly by potently
enhancing spore formation at ecologically relevant concen-
trations in the farmer strain, and suppressing spore formation in
the nonfarmer strain. The other, pyrrolnitrin, showed similar effects,
and it is likely to counter microbial pathogens of other species.
The farmer strain was resistant to the inhibitory effects of pyr-
rolnitrin and chromene, which indicated that the farmer had
adapted to carrying the nonfood strain PfA. Chemically un-
characterized supernatants collected from different non-food
bacteria carried by other D. discoideum clones also help their
farmer clone and harm non-carrier clones (37). Taken together,
these observations provide a strong argument for coevolution of
both the farmer and symbionts; they also establish that the sym-
biosis is not just a food and farmer symbiosis, but rather displays
some of the multipartite qualities of other farming symbioses, such
as the fungus-farming ants and bark beetles with their crop fungi
providing the food and the bacterial symbionts providing chemical
defenses against fungal pathogens (5, 7–9). What distinguishes this
farming symbiosis is the close relatedness of PfB and PfA—the
bacterial food source and the small-molecule producer (although
this closerelatednessdoesnotholdforbacterialsymbionts ofother
Dictyostelium farmers). A single point mutation in the gacA gene
is sufficient to completely alter the chemical repertoire of the
nonfood source, thus effectively changing its role in the symbiotic
association from secondary metabolite producer to food source.
Finally, these findings highlight the usefulness of investigating
the small-molecule chemistry that underlies so much of bacte-
rial–eukaryotic symbiotic associations as a source of both new
chemistry (molecules and pathways) and biology (defense, de-
velopment, cooperation, and evolution).
Materials and Methods
P. fluorescens and D. discoideum Growth. We grew both P. fluorescens strains
at 30 °C in SM/5 liquid media containing 2 g D-glucose, 2 g Bacto Peptone
(Oxoid), 2 g yeast extract (Oxoid), 0.2 g MgCl2, 1.9 g KHPO4, and 1 g K2HPO4
per liter. We grew the farmer (QS161) and nonfarmer (QS160) Dictyostelium
discoideum from spores on SM/5 agar plates (SM/5 liquid media supple-
mented with 15 g agar per liter) in association with Klebsiella pneumoniae
as bacterial food source, at room temperature (21 °C) in Petri dishes (100 ×
15 mm). For cocultures, we grew the farmer QS161 in association with its
bacterial symbionts PfA and PfB (in a 1:10 ratio). For the complementation
assay, we grew the farmer QS161 in association with PfB and 200 μL of cell-
free PfA supernatant.
LC-MS Analysis of Bacterial Secondary Metabolites. A 1-L culture of the re-
spective bacterial strain in SM/5 media was grown for 24 h at 30 °C and 200
rpm. After 24 h, we extracted 15 mL of the culture with 25 mL ethyl acetate
(EtOAc). We separated the organic phase, dried it over Na2SO4, and concen-
trated it in vacuo. The cocultures were soaked with EtOAc, and the organic
phase was separated by filtration and concentrated in vacuo. The crude con-
centrates were redissolved in 15% (vol/vol) methanol in water and applied to
a Waters C18 Sep-Pak column (0.5 g) and washed with 15% (vol/vol) acetonitrile
in water to remove polar molecules. The column was eluted with 100%
acetonitrile, and solvents were removed in vacuo to yield a brown concentrate.
The concentrates were then subjected to HPLC-MS analysis using an
Agilent HPLC system equipped with a diode array detector, and a 6130 Series
quadrupole mass spectrometer was used with a Phenomenex Luna C18 (5 μm,
100 Å 100 × 4.6 mm, flow rate = 0.7 mL/min) column. We used the following
gradient for HPLC-MS analysis: 0−1 min, isocratic 30% acetonitrile in water +
0.1% formic acid; 1−21 min, linear gradient from 30% acetonitrile in water +
0.1% formic acid to 100% acetonitrile + 0.1% formic acid.
Spore Assay. We prepared log-growth amoeba by plating 2 × 105spores of
each clone in association with K. pneumoniae on SM/5 agar plates. Amoeba
log growth occurs ∼32–36 h after plating spores. At this time, we collected
amoebas and washed them free of bacteria using ice-cold aqueous KK2
buffer (2.2 g KH2PO4and 0.7 g K2HPO4per liter). We determined amoeba
density with dilution using a hemacytometer and a light microscope. For the
filter-pad assay, we prepared dilutions of either chromene or pyrrolnitrin in
KK2 + 0.5% DMSO from stock concentrations prepared in 100% DMSO. We
used 150 × 15 mm Petri plates lined with two layers of Whatman no. 3
(Schleicher & Schuell) soaked with either control buffer or the various test
dilutions laid with a grid of equidistant 13-mm square AABP 04700 (Millipore)
black filter squares. To test social-stage spore production, we spotted the
filters individually with 1.25 × 106amoebas in starvation buffer, and we
prepared duplicate samples for each clone for each experiment. We allowed
the clones to hatch, grow, and develop under direct light to limit potential
movement of slugs before final culmination to fruiting bodies. Fruiting body
formation for all clones was complete after ∼24 h. We allowed the spores to
mature in the fruiting bodies for an additional 24–48 h before collection. To
determine spore number, we placed each test filter with fruiting bodies in
an Eppendorf tube containing 1.0 mL KK2 + 0.1% Nonidet P-40 alternative
(Calbiochem). The tubes were vortexed briefly to disperse the spores, and
beneath branches show the branch length, and the numbers by the node
show the bootstrap value of 500 replicates. Note that branch lengths are not
drawn to scale and are very short in the bottom clade of five strains.
Maximum-likelihood tree using 20 conserved genes. The numbers
| www.pnas.org/cgi/doi/10.1073/pnas.1308199110Stallforth et al.
the spores were counted without dilution using a hemacytometer. We cal-
culated spore number for experimental treatments as a percent change
compared with control based on spore number recovered from starvation
buffer control samples.
Sequencing. Genomic DNA of PfA-QS161, PfB-QS161, Pf-QS68, and Pf-QS152
was obtained from a 5-mL overnight culture at 30 °C in SM/5. Extraction of
DNA was performed using GenElute Bacterial Genomic DNA Kit (NA2100;
Sigma Aldrich). Next Generation Illumina sequencing with 100-bp single-end
reads was performed at Harvard Medical School Biopolymers Facility. Reads
were mapped to the reference genome sequence of Pf5 using Burrows–
Wheeler Aligner software with default parameter settings (38). The SNPs
and consensus genome sequences were called using SAMtools (39). A max-
imum-likelihood tree was constructed from concatenated DNA sequences of
20 genes (dnaG, gcp, infB, ksgA, nusA, nusG, rplA, rplC, rplE, rplF, rplK, rplN,
rpoB, rpsB, rpsC, rpsD, rpsG, rpsH, secY, and ychF), a subset of the topologically
congruent genes as reported by Bapteste et al. (40), from newly sequenced
strains and other P. fluorescens strains with completed genome sequences
that are retrieved from NCBI’s BioProjects database (www.ncbi.nlm.nih.gov/
bioproject/browse/)usingthe HKYmodel byMEGA5(41). P. syringaepv. tomato
strain DC3000 was included as an outgroup. We used DNA sequences rather
than amino acid sequences because the group including the four D. discoideum
carried clones, and Pf-5 differed by only one amino acid in these 20 genes.
Construction of Deletion Mutants in P. fluorescens PfA-QS161. Antibiotics were
used in culture media at the following concentrations: gentamicin 15 μg/mL
for E. coli and for P. fluorescens. To counterselect E. coli donor cells in gene-
replacement experiments, nalidixic acid was used at a concentration of
15 μg/mL; mutant enrichment was performed with gentamicin at a final
concentration of 15 μg/mL. Small- and medium-scale preparations of plasmid
DNA were made with the QIAprep Spin Miniprep kit (Qiagen, Inc.). PCR was
performed using a KOD hot start polymerase kit (Novagen, EMD Millipore).
DNA fragments were purified from agarose gels with QIAquick Gel and PCR
Purification Kit (Qiagen, Inc.). All constructs obtained by PCR techniques
were confirmed by sequence analysis (Genewiz). A clean gacA deletion
mutant of P. fluorescens PfA-QS161 was constructed using the suicide plas-
mid pEXG2 (42). Primer sequences and a detailed description of cloning
procedures are provided in SI Appendix.
ACKNOWLEDGMENTS. We thank Cara Haney, Taehyun Kim, and Steve Lory
forhelpfuldiscussions, vectors,andplasmids;Anna-BarbaraHachmannfor help
with the sequence alignments of the P. fluorescens genomes; and Jeanne Salje
for taking EM photographs of PfA and PfB for Fig. 1. This work was supported
by a Swiss National Science Foundation and Feodor Lynen Postdoctoral Fellow-
ship (to P.S.), National Institutes of Health Grant GM086258 (to J.C.), and
National Science Foundation Grants DEB1204352 and DEB1201671 (to D.C.Q.
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