Tangled bank of experimentally evolved Burkholderia
biofilms reflects selection during chronic infections
Charles C. Traverse, Leslie M. Mayo-Smith, Steffen R. Poltak, and Vaughn S. Cooper1
Department of Molecular, Cellular, and Biomedical Sciences, University of New Hampshire, Durham, NH 03824
Edited by James M. Tiedje, Michigan State University, East Lansing, MI, and approved November 29, 2012 (received for review April 26, 2012)
How diversity evolves and persists in biofilms is essential for
understanding much of microbial life, including the uncertain
dynamics of chronic infections. We developed a biofilm model
enabling long-term selection for daily adherence to and dispersal
from a plastic bead in a test tube. Focusing on a pathogen of the
cystic fibrosis lung, Burkholderia cenocepacia, we sequenced
clones and metagenomes to unravel the mutations and evolution-
ary forces responsible for adaptation and diversification of a single
biofilm community during 1,050 generations of selection. The mu-
tational patterns revealed recurrent evolution of biofilm specialists
from generalist types and multiple adaptive alleles at relatively few
loci. Fitness assays also demonstrated strong interference compe-
tition among contending mutants that preserved genetic diversity.
Metagenomes from five other independently evolved biofilm lin-
eages revealed extraordinary mutational parallelism that outlined
common routes of adaptation, a subset of which was found, sur-
prisingly, in a planktonic population. These mutations in turn were
surprisingly well represented among mutations that evolved in
cystic fibrosis isolates of both Burkholderia and Pseudomonas.
These convergent pathways included altered metabolism of cyclic
diguanosine monophosphate, polysaccharide production, tricar-
boxylic acid cycle enzymes, global transcription, and iron scaveng-
ing. Evolution in chronic infections therefore may be driven by
mutations in relatively few pathways also favored during labora-
tory selection, creating hope that experimental evolution may
illuminate the ecology and selective dynamics of chronic infections
and improve treatment strategies.
population genomics|microbial ecology|ecotype|clonal interference
may involve adaptation to life in biofilms. These aggregations of
cells on surfaces are more spatially structured and both generate
and experience more diverse environmental conditions than
well-mixed planktonic populations, leading to increased bio-
diversity (1–4). Biofilm diversity often evolves from founding
clones and commonly is observed as variation in colony mor-
phology, production of extracellular polymers, motility, and se-
creted signals or substrates (3, 5, 6). Because isolates recovered
from chronic infections of different patients often resemble these
biofilm-specific variants (7–9), they may reflect adaptation to
similar selective forces in vivo. However, the nature of selection
during infections and why multiple types evolve is uncertain;
despite several recent surveys of evolution in the lungs of CF
box. For this reason, we developed a model enabling long-term
selection in biofilms (4) to study how genetic and ecological di-
versity evolves in a structured environment.
Our simple model involves a daily cycle in which cells must
form a biofilm on a plastic bead suspended in minimal medium
in a test tube (Fig. 1). Colonized beads then serve to inoculate
the next tube containing a fresh bead, so bacteria must remain
adherent during transfer and then disperse to colonize a new
bead. In this environment, we studied adaptation by a soil isolate
of Burkholderia cenocepacia, an opportunistic pathogen responsible
for fatal biofilm-associated lung infections in CF patients (14, 15).
acterial evolution during chronic infections, particularly in
pulmonary infections of persons with cystic fibrosis (CF),
Although this species has been identified as the most prevalent and
pathogenic of the Burkholderia cepacia complex species in CF
patients (14), relatively little is known about how it adapts to
a biofilm lifestyle or to the CF lung environment (5, 16).
Six B. cenocepacia populations underwent ∼1,050 generations
of biofilm selection, all of which diversified into similar sets of
three distinct, heritable colony morphotypes from a single an-
cestral clone (Fig. 1) (4). At least one of these morphotypes—
a small, highly rugose “wrinkly” type (17)—is commonly isolated
from chronic lung infections of both Burkholderia spp. and
Pseudomonas aeruginosa and has been associated with high pa-
tient mortality (7, 17). Each morphotype associates with a dis-
tinct ecological niche and thus can be considered an ecotype;
collectively, ecotypes segregate nutrients and biofilm space in
ways that enhance community productivity (Fig. 1D) (4). The
persistence of these ecotypes for the duration of the experiment
raised several questions about their evolutionary dynamics and
mutational causes. Did each type evolve once and persist, or did
multiple mutations produce the same ecotypes? In addition, did
these types generate further diversity and coevolve, much as
Darwin envisioned for the evolution of his famous “tangled bank”
(18) of myriad, interdependent plants and animals?
We also sought to test whether mutations favored during pro-
longed selection in experimental biofilms occurred in targets similar
to those identified during genetic surveys of CF lung isolates, in-
cluding small-colony variants. We applied multiple, complementary
genomic approaches to both clones and communities to identify the
molecular bases of biofilm adaptation and quantify mutant frequen-
cies over time. These results allowed us to reconstruct a nearly com-
in one biofilm population with unprecedented resolution. Compar-
ing these mutations with those found in five other replicate experi-
genes known to affect biofilm production and in unexpected path-
ways. Moreover, many of these mutations occurred in genes that
commonly are mutated among isolates of Burkholderia and
P. aeruginosa fromchroniclunginfections,implyingthatadaptation
This study focused on a single biofilm population [B1, from
a larger experiment (4)] in which three distinct colony morphol-
Author contributions: C.C.T. and V.S.C. designed research; C.C.T., L.M.M.-S., S.R.P., and
V.S.C. performed research; C.C.T. contributed new reagents/analytic tools; C.C.T., L.M.M.-S.,
S.R.P., and V.S.C. analyzed data; and C.C.T. and V.S.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.
Data deposition: The sequence reported in this paper has been deposited in the National
Center for Biotechnology Information Short-Read Archive, http://www.ncbi.nlm.nih.gov/sra
(accession no. SRP004277), in accordance with Joint Genome Institute policy.
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.
www.pnas.org/cgi/doi/10.1073/pnas.1207025110PNAS Early Edition
| 1 of 10
ogies that correspond with unique ecological functions evolved:
studded (S), ruffled (R), and wrinkly (W) (Fig. 1C). The S mor-
photype is the most abundant and retains the capacity to grow
planktonically while forming thin, mostly confluent biofilms;
therefore it can be considered a generalist. The R morphotype is
a specialist and produces more copious biofilm and fewer plank-
tonic cells. The rarest W type primarily grows as a dense, towering
differences, we sequenced DNA from mixed communities (met-
agenomes), the complete genomes of representative clones, and
alleles of 60 alternative clones from multiple time points. These
analyses (Table 1) revealed that early ecotypes (isolated at gen-
eration 315) evolved independently by distinct mutations, whereas
late ecotypes (isolated at generation 1,050) derived from a com-
mon lineage and subsequently evolved by one to four ecotype-
specific mutations. Thus, although the composition of the com-
munity—a majority S ecotype and minority R and W ecotypes—
remained constant, its genetic structure changed and suggested
that R andW types hadevolved anewfrom anShaplotype.Within
the S lineage alone, five mutations became detectable within ∼315
mL (the minimum number of cells adhering to the bead that was
transferred). Given this large population size and the fact that the
mutation rate did not evolve (4), genetic drift cannot explain the
rapid rise of these mutations. Rather, the following features
Mutations in coding sequences were mostly nonsynonymous
(nonsynonymous/synonymous ratio = 5.5), intergenic mutations
were associated with likely promoters, and deletions affected
genes that were plausible targets of selection (Table 1). The rapid
rise of these lineages combined with the low per-genome muta-
tion rate (<10−2per genome per generation) also theoretically
should hinder hitchhiking of neutral mutations but does not ex-
clude this possibility.
Phylogeny and Ecological Dynamics of Biofilm Diversification. To
determine how well the fully sequenced clones represented the
ecotype populations, and to evaluate the accuracy of mutation
read depth in the metagenomes as estimates of allele frequency,
10 clones of S, R, and W from the early and late communities
were screened for key mutations (Table 1). Read depth in the
metagenomes correlated well with mutation frequency among
screened clones (linear regression: F = 567.5, df =1, P < 0.0001,
r2= 0.99). Between 10 and 20 mutations were reliably detectable
at any given time, and perhaps more if lower-quality reads are
considered (Table 1, and Table S1). Importantly, sequencing
individual clones linked mutations in the metagenome to certain
ecotypes and haplotypes (e.g., M14 and M18 defined new W
lineages), and these screening data (Tables S2 and S3) enabled
a stepwise, most parsimonious phylogeny of this biofilm pop-
ulation (Fig. 2). By combining this phylogeny with estimates of
allele frequency, we developed a model of how the genetic and
ecological composition of the community evolved throughout
the experiment (Fig. 3). The competitive dynamics of genotypes
acting both within and between niches became more apparent
diversification. (A) B. cenocepacia HI2424 was grown in GMM with a 7-mm
polystyrene bead for 24 h. This bead was transferred to a new tube of GMM
with an oppositely marked bead. Cells that adhered to the first bead needed
to disperse and attach to the oppositely marked bead, which then was
transferred to another tube after 24 h of growth. These bead-to-bead
transfers were conducted for ∼1,050 generations. (B) After ∼300 generations
Summary of the biofilm experimental evolution and its ecological
of biofilm selection, an adaptive radiation of biofilm generalists (S) and
specialists (R and W) was observed. Subsequently, each ecotype remained
detectable throughout the experiment. (C) Each ecotype has distinct growth
patterns when grown in monoculture. S occupies the planktonic phase and
displays moderate bead attachment, R exhibits less planktonic growth than
S and forms thick biofilms, and W forms clumps and produces copious
amounts of biofilm. (D) Ecotypes vary in biofilm production (3); however,
when S, R, and W are grown together, total biofilm is greater than expected
from their monoculture values and starting frequencies, indicating synergy
when all ecotypes are present (Methods). Assays were focused on sequenced
clones isolated at 1,050 generations; error bars indicate the 95% confidence
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and suggested a more fluid community structure. In addition,
new mutants coexisted with their ancestors for hundreds of
generations, in contrast to the sequential replacement that often
occurs in unstructured, homogeneous environments. Each eco-
type also remained genetically diverse, often by different muta-
tions in genes of similar putative function.
The S type was the first to achieve high frequency and, as
suggested by the genome of an early S clone, evolved by se-
quential mutations that eventually became globally successful.
The dominant S lineage originated with an SNP in yciR (M1),
which encodes a protein with three domains: a PAS-sensor,
a GGDEF diguanylate cyclase, and an EAL phosphodiesterase
(19); this mutation therefore likely affected metabolism of cyclic
diguanosine monophosphate (c-di-GMP). Shortly thereafter,
a second SNP (M2) occurred 27 genes downstream from this
initial mutation on the same haplotype, and this lineage in-
creased to ∼95% of the population (Fig. 3). However, before this
lineage could fix, a different yciR SNP occurred and produced
the first R ecotype (M15). Similarly, another SNP in a homolog
of wspA (M16) occurred in association with the first detected W
ecotype. The wsp operon has been characterized extensively in
Pseudomonas species (20, 21) for its production of a wrinkly
spreader phenotype related to c-di-GMP metabolism. These
mutations imply that varied c-di-GMP metabolism was the cause
of ecological differentiation and set the stage for prolonged co-
existence of S, R, and W (Fig. 3B).
Mutations on the S lineage continued to accumulate but also
began to produce new R and W lineages (Fig. 3B). The next S
mutation was an SNP in the tricarboxylic acid (TCA) cycle in-
termediate enzyme, 2-oxoglutarate dehydrogenase (OGDH, M3),
which displaced its direct ancestor and also produced a new R
variant (Fig. 3B). Then a peculiar set of mutations (M4 and M5)
followed in an uncertain order: M4 consisted of a two-codon de-
letion in a transcriptional regulator, and M5 was a large deletion
that removed the mutated yciR locus and the new M1 and M2
alleles. This new M3/M4/M5 haplotype also spawned a successful
R type. Because this newer haplotype displaced earlier S hap-
lotypes, the frequencies of M1 and M2 declined but remained
detectable (Fig. 3A). A single base-pair deletion (M6) in manC,
a gene associated with exopolysaccharide (EPS) and LPS pro-
duction, occurred next in this dominant lineage and then gave rise
to yet another new R and a W variant. These niche invasions in-
volved three new R lineages and one new W lineage (Fig. 3C; the
mutations responsible for these invasions are unknown) that drove
the original R resident (M15) extinct but did not noticeably affect
the original W lineage. The early W lineage also had acquired two
new mutations (M17 and M18). The R and W biofilm specialist
ecotypes therefore evolved recurrently from a single, dominant
The final defining mutation of the dominant lineage (M7) was
the most successful mutation in the experiment and altered the
promoter of the iron-storage gene bacterioferritin. Remarkably,
M18 that evolved on a W haplotype altered the same promoter
(Table 1); the dynamics during this period suggest that M7 may
have evolved in response to M18. M18 was detected between 315
and 525 generations and was associated with a large gain in W
frequency (increase in red fraction in Figs. 3 and 4) to ∼30% of
the entire population. Subsequently, the rise of M7 in the main S
lineage associated with the decline of W ecotypes to their orig-
inal ∼10% (decline in red fraction at 600 generations, Figs. 3 and
4). Coincidentally, all R types lacking a bacterioferritin mutation
went extinct, and only new R mutants evolving from the M7
haplotype (e.g., M10) preserved this ecotype. However, the fre-
quency of the W lineage with mutation M18 apparently did not
change when two new W lineages invaded from the M7 haplo-
type later in the experiment (Fig. 3), presumably because each of
these lineages had a similar bacterioferritin mutation. Once
again, the new W types evolved by single mutations in wsp
homologs, wspA (M13) and wspE (M14), similar to the original
W lineage. These dynamics highlight the global importance of
acquiring a mutation in the bacterioferritin promoter sequence
and the capacity for small-colony variants to re-evolve.
their predicted functional significance
Mutations detected in one population during 6 mo of selection under a daily cycle of biofilm colonization and dispersal and
(in generations)Locus AnnotationMutational effectSignificance
Transcriptional regulator 5′ to
yciR and 94 genes downstream
Del 38, del 39, L40V
S, 315 S,R,W, 1050
S, 315 S,R,W, 1050
M5S, 315 S,R,W, 1050 Del, 95 genes, including
M1 & M2
STOP @ 263
−10 G → A promoter
del, 49 genes
−10 G → A promoter
−35 G → A promoter
S, 315 S,R,W, 1050
5′ of bacterioferritin
Loss of 49 diverse genes
5′ of mltA
5′ of bacterioferritin
Ferrochelatase (in ancestor)
Altered RNAP affinity
Ecotypes (S, R, and W) are defined as in Fig. 1, and if the mutation is associated with a particular time point, it was identified in a fully sequenced isolate.
Most mutations (bold font) were subsequently screened in multiple isolates using Sanger sequencing.
Traverse et al.PNAS Early Edition
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By retrospective sampling of the population throughout its
evolution, we found that the relative frequency of each morpho-
type, and hence the balance of their respective ecological strate-
gies, indeed fluctuated over time (Fig. 4). Shifting morphotype
frequencies were tightly paired with changing allele frequencies,
most often when new mutants of the S lineage invaded and dis-
placed existing S, R, or W inhabitants (Fig. 3). This pattern of
ecological and genetic invasion from the numerically dominant S
niche into the more specialized R and W niches suggests that
high-biofilm specialists evolve from generalists, but not vice versa.
Therefore the highly productive, synergistic late community (Fig.
1D) (4) mostly comprises ecotypes that are immediate genetic
relatives with only one or two mutations defining them.
Clonal Interference Within and Among Niches.Each ecotype remained
genetically diverse throughout the experiment (Fig. 3), indicating
that beneficial mutations were relatively common and likely
competed with one another (22). This dynamic of clonal inter-
ference can effectively reduce the relative benefit of any single
mutation or haplotype and may allow multiple lineages to per-
sist. Theory suggests that such competition also can produce a
simultaneous sweep of multiple linked mutations (e.g., mutations
M3–M6, Fig. 3A), because more than one beneficial mutation
may be required to prevail in competition (22, 23).
We began to quantify the selective forces acting on each
mutational step leading to the dominant haplotype by compe-
tition between these mutants and two reference clones: with the
founding ancestor and with a highly adapted intermediate S
clone (Table 2). Competition versus the original ancestor pro-
vides a common reference to judge fitness in the absence of
other beneficial variation, whereas competition versus a well-
adapted “contemporary” mutant illustrates selective forces among
contending adaptive lineages. Each mutant displayed fitness
advantages that would displace the ancestor before 70 generations
in the absence of clonal interference; in actuality, none of these
alleles fixed, and their frequencies increased approximately 10-fold
more slowly in the evolving community (Table 2). However, the
slight variation in fitness among mutants versus the ancestor did
not explain how selection favored the mutants’ rapid succession. In
contrast, far greater fitness differences among haplotypes were
evident from competitions with the most derived (M3–M7) hap-
lotype, which was considerably fitter versus an early-evolved
mutant than versus the original ancestor. The fitness ranks of
evolved haplotypes were consistent with the mutational order
and demonstrated a large advantage of the yciR deletion (M5).
Evidently, selection in this biofilm system is strongly context
dependent, favoring mutants that are superior in both the current
biotic environment comprising multiple lineages and in the fluc-
tuating abiotic conditions.
Nonetheless, the extraordinarily high fitness advantages of
evolved mutants in pairwise competition far exceed their realized
benefits in the community, as judged either from the population
dynamics of the entire metagenome or among alleles within
a niche (Table 2). The fact that many contending adaptive lin-
eages are found at appreciable frequencies throughout the ex-
periment indeed may have diminished the net advantage of any
given haplotype. In addition, more complex frequency- or den-
sity-dependent interactions among favored genotypes, such as
the cross-feeding interactions described previously (4), may have
slowed the rise of adaptive alleles and preserved genetic diversity
within the biofilm community.
Genetics and Physiology of Biofilm Adaptation. The functional sig-
nificance of the mutations associated with adaptation to pro-
longed biofilm selection highlights the challenges faced by
Burkholderia in this system and the changes that were favored.
Four major pathways of adaptation emerged, described below,
but some mutations do not fall into these categories, suggesting
that selective forces in this biofilm system are manifold and may
preserve many alternative alleles.
Altered regulation of c-di-GMP. Three yciR mutations (M1, M5, and
M15)werecritical tothe earlyevolution ofthebiofilm community
(Fig. 3). M1 (Y355D) occurred in a highly conserved residue of
the GGDEF domain and was associated with S morphology.
M15 (A106P) was found in the PAS sensor domain of yciR and in
isolation defined R morphology; thus, alternative yciR alleles
defined morphological and ecological differences in this system.
The third yciR mutation (M5) deleted this locus and 94 other
genes and was likely mediated by an IS116-family insertion se-
quence at the 3′ end of this lesion. This mutation was also asso-
ciated with S morphology, but its ecological dominance over the
two yciR SNPs remains to be explored. Curiously, disrupting this
gene in another Burkholderia species had been shown previously
to decrease both biofilm production and quorum sensing (24).
In general, wrinkly colony morphologies nearly always are
associated with increased c-di-GMP concentrations and in
Pseudomonas may be caused by mutations that lead to auto-
induction of an associated diguanylate cyclase enzyme, WspR
(21). Four nonsynonymous mutations were detected in this op-
eron, each in different clones, including two in wspA (M13 and
M16) and one each in wspD (M12) and wspE (M14). All wsp
mutations were associated with either an R or W morphotype:
wspA and wspE alleles were found in W clones, and wspD was
found in the successful R lineage (Fig. 3B). The wspE mutation
occurred in an active site for phosphorylation and hence is likely
to alter signaling of this pathway. Presumably, each of these four
mutations adaptively alters the levels of c-di-GMP in this system,
a subject of ongoing study, and also defines particular ecological
roles and leads to niche subdivision.
(Not to scale)
population. Haplotypes were assembled by screening 30 clones from 525
generations and 30 clones from 1,050 generations at variable loci in the
metagenome sequences of the community, and their most parsimonious
phylogeny was constructed. The ancestor (black line) is rapidly displaced by
new mutant ecotypes depicted in different colors; lineages that do not
continue were outcompeted by superior lineages within their niche. “Un-
known” lineages are ecotypes of uncertain genetic composition, i.e., the
mutation defining the R or W morphology is unknown.
Phylogeny of adaptation and diversification in a model biofilm
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Shifts in central metabolism. Three beneficial mutations, M2, M3,
and M4 (Tables 1 and 2), likely altered central metabolism. M2 is
a mutation in a gene annotated as FAD-binding monooxygenase.
This type of enzyme may be involved in electron transport, and
altering the levels of FAD(H) within the cell may provide
a growth advantage in the selective environment. M3 is a non-
synonymous mutation in OGDH (M3), and M4 is a two-codon
deletion from a LysR-like transcriptional regulator that is 5′ to
a gene encoding lactate dehydrogenase (M4). These mutations
could enhance growth in galactose minimal medium, but they
also may reflect selection for biofilm production.
Altered mannose metabolism related to EPS and LPS production and
adherence. The globally successful mutation M6 created a pre-
mature stop codon in a multidomain gene (manC) that is in-
volved in mannose metabolism and is located within a gene
cluster related to LPS biosynthesis. The gene is bifunctional, with
an N-terminal mannose-1-phosphate guanylyl transferase do-
main and a C-terminal mannose-6-phosphate isomerase (PMI)
(25). A single-nucleotide deletion occurred in a poly-A tract (a
relatively rare sequence in this genome, given its 67% GC con-
tent) that preserved the N-terminal domain but truncated the
C-terminal PMI domain and likely generated polar effects on the
rest of the operon. The effects of this disruption are potentially
widespread, because downstream genes are expected to participate
in LPS biosynthesis. Late S, R, and W clones produced less biofilm
when a functional manC was added on a plasmid (Fig. S1). We
hypothesize that this mutation effectively directs mannose toward
exopolysaccharide and away from energy production and may in-
crease adherence and coherence as a response to disrupting LPS
biosynthesis, as has been reported elsewhere (26, 27).
Enhanced iron competition. The two different mutations in the bac-
terioferritin promoter sequence (M18 and M7) occurred in the −35
and −10 σ factor-binding domains, respectively. Quantitative RT-
PCR of genotypes with the M7 mutation exhibited increased tran-
scription at this locus (Fig. S2), indicating that these mutants gained
capacity for iron storage. Because the experimental environment
was not supplemented with iron, this resource likely became limit-
ing. Another iron-associated mutation evolved in the late commu-
nity in a gene encoding ferredoxin (Table S1), providing further
evidence of selection for altered iron metabolism. Thus, competi-
tion for iron occurred both within and between niches in the biofilm
community, which favored mutants with either M7 or M18, dis-
placed mutants lacking these alleles, and facilitated invasion from
the S niche to the R and W niches.
Evolutionary Parallelism Among Six Independently Evolved Biofilm
Populations. Although we have not assembled a detailed evolu-
tionary model of all six biofilm populations (as in Fig. 3), mutations
Ecotype Niche Breadth
lines), and dynamics were interpolated. (A) Frequency of majority mutations belonging to the dominant haplotype throughout the community. (B) Mutational dy-
namics within and among niches. Each color transition represents a new haplotype (labeled as in Table 1), and color breadth shows haplotype frequency in the
community, to scale. The earliest mutants arose on the ancestral genotype, and subsequent mutations evolved within the ecotypes that subdivided the community.
Lines crossing ecotype boundaries (light blue lines) represent invasion of the dominant S haplotype into the R or W niche associated with novel mutations. Horizontal
light blue lines highlight the ecological boundaries that evolved within this community. Additional low-frequency mutations were detected in the metagenomes and
are reported in Table S1, and other mutants likely evolved before the first samples. *, R isolate with unknown niche-specifying mutation; **, W isolate with unknown
niche-specifying mutation. (C) Dynamics of niche invasions by mutants of S over time. Each blue arrow represents the invasion of an S type into an R or W niche.
Population genomics and ecological structure of the biofilm community over time. Allele frequencies were determined at four time points (vertical dashed
Traverse et al.PNAS Early Edition
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associated with adaptation were identified in the early and late
samples of all populations. Exceptional parallelism among
adaptive targets occurred: all six populations experienced muta-
tions in the four major functional categories outlined previously
(Table 3 and Table S4). At least 26 mutations involved in c-di-
GMP metabolism occurred, including seven independent muta-
tions in the yciR gene, 18 independent mutations in the wsp op-
eron, and one in an unclassified diguanylate cyclase. Another
commonly mutated area across the populations was a gene cluster
involved in LPS biosynthesis, including mutation M6. Twenty
independent mutations occurred within this gene cluster among
all six populations, thus suggesting that altered LPS may be
adaptive in biofilms. Mutations in OGDH also were successful in
each population, highlighting this step in the TCA cycle as limiting
for the ancestor in this environment. Other classes of mutations
occurred in replicate populations as well, including in RNA
polymerase subunits rpoC and rpoD (n = 5) and in a galactose
metabolism operon (n = 11).
A population evolved under planktonic conditions in the same
medium, selected because it was the only one of six to evolve an
additional colony type, was sequenced also. This population also
revealed some parallelism with the biofilm lines, including three
mutations in the LPS gene cluster, two mutations in rpoC, and
one mutation in yciR (Table S5). These results imply that ad-
aptation to the high-galactose environment lacking the bead (but
preserving all other conditions, including constant 37 °C and the
potential for biofilm growth on the test tube walls that may have
been shed and preserved during transfer) may explain some of
these alleles. Alternatively, different alleles in the same gene
may be favored by planktonic or biofilm growth. However, none
of the wsp mutations associated with R and W types were
detected, and three unique mutations in the quorum-sensing
regulator cepR (28) evolved in this planktonic population. In
general, the high genetic parallelism in adaptive targets sug-
gested that adaptation in this model proceeded along relatively
few pathways, despite progressive ecological complexity and the
ease of selecting adaptive mutants (which evolve in a matter of
days) from the ancestral clone in this system.
Convergent Evolution with CF Lung Isolates. The same four classes
of mutations found in our replicate populations were found in
studies of isolates of Burkholderia dolosa and Pseudomonas aer-
uginosa that evolved during CF infections (Table 4) (10–12). We
make this comparison cautiously, because more mutations were
reported in these clinical studies than in this system and because
database matches may be uncertain. However, the degree of
overlap for these functions is remarkable and is statistically im-
probable to have occurred by chance alone, given the number of
observed mutations in each study and the number of genes as-
none of the mutations that uniquely evolved in the planktonic
six independent wspF mutations in their Pseudomonas aeruginosa
study, which highlights altered c-di-GMP regulation in certain
infectious lineages. Another study of P. aeruginosa reported two
more mutations in this operon, wspE and wspC (Table 4) (11),
although these mutations could be explained by chance. Eighteen
distinct mutations in the wsp operon were selected among our
replicate populations. In addition, five OGDH mutations were
detected in 112 clinical B. dolosa isolates, suggesting the impor-
tance of altered TCA cycle enzyme activity. Likewise, OGDH
mutations evolved in all six biofilm populations and were associ-
ated with increased fitness in population B1.
One of the more remarkable examples of convergent evolu-
tion involved a gene cluster responsible for synthesizing O-an-
tigen LPS, in which manC (mutation M6) is found. Lieberman
et al. (10) identified 19 mutations in this locus across 112 clinical
isolates and one additional mutation in another manC homolog.
Among the six experimental biofilm populations studied here, 20
independent mutations in the homologous and well-conserved
gene cluster were found. Mutations in manC evolved in both B.
dolosa (n = 2) and B. cenocepacia (n = 4). The remainder of the
mutations occurred in similar but not identical sugar transferase
genes in both organisms. Similarly, LPS-associated mutations
have been detected in clinical P. aeruginosa isolates in the wbpA,
wbpW (a manC homolog), and wbpZ genes (11, 29). Given this
exceptional mutational parallelism found among relatively few
studies, it is likely that some of the same forces that drive biofilm
adaptation also contribute to adaptation of these pathogens
within the CF lung.
from plate counts. As certain mutations became detectable (arrows and
mutation labels), changes in morphotype frequencies were observed.
Ecotype frequencies in biofilm population B1 over time as evaluated
Table 2.Fitness estimates of evolutionary steps within the dominant S haplotype
Evolved S haplotypeFitness vs. ancestor Fitness vs. M3-M7 Fitness from whole metagenomeFitness from niche metagenome
Haplotypes are defined by mutations listed in Table 1. Empirical fitness (columns 1 and 2) was calculated by direct competitions between clones (column 1,
5–9 replicates, column 2, 11 or 12 replicates) and is reported as selective rate constants with 95% confidence intervals in parentheses. Haplotypes vary
modestly in competition versus the ancestor (F = 4.38, P = 0.0058) but vary widely in their fitness versus the most derived genotype (F = 46.0 P < 0.0001),
demonstrating very strong selection among co-occurring mutants. Much lower realized fitness differences were inferred from metagenomes (columns 3 and
4) as changes in mutation frequency over time, either throughout the community (column 3) or within niches associated with ecotypes (column 4).
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Adaptive diversification and niche partitioning have been ob-
served previously in bacterial populations that were evolved ex-
perimentally in structured environments (30–33), so the evolved
diversity in this biofilm model is unsurprising. However, we lack
understanding of the population genetic dynamics of biofilm
communities over the longer term and particularly how newly
established lineages might coevolve and shape their environ-
ments. Further, to our knowledge, experimental selection for the
ability of bacteria to both attach and disperse has not been
performed. Our previous observation that three distinct ecotypes
evolved and persisted in each of six replicate evolved biofilm
populations (4) led us to anticipate that each type represented
a distinct lineage that had evolved continuously by endemic
alleles. We also found that ecotype frequencies varied over time,
and biofilm communities became more productive (4), suggesting
that the ecotypes had coevolved. However, a detailed population
genomic analysis of one biofilm community, made possible by
Table 3.Mutations shared among the six evolved biofilm populations, categorized by putative function
FunctionLocusAnnotation No. of mutations
5′ of galactose metabolism operon
iciR family (regulator of galactose metabolism)
dTDP-glucose 4,6-dehydratase (3′ of manC)
fkbM, methyltransferase (5′ of manC)
Glycosyl transferase, family 2
capB, polysaccharide synthesis
GGDEF, PAS/PAC Sensor
LPS gene clusterYP_834517
Parallel evolution in each functional category occurred much more often than expected by chance (Table S6). The full list of mutated
positions is found in Tables S1 and S4. NC, noncoding.
*One mutation is the likely polar effect of a deletion and premature stop in the gene found 5′ in the operon.
reported for B. dolosa (10) and P. aeruginosa (11, 12) and mutations that evolved in this experimental system (Table 3)
Convergence among mutations found in isolates from chronic pulmonary infections of CF patients, as
B. dolosaAnnotationMutationsP. aeruginosa AnnotationMutations
LPS gene cluster BDAG_02317
10 2 (ns)
1 (ns)6wbpW (manC)
PA3159wbpA 1 (ns)
Cyclic di-GMP1 (ns)PA14_16450
Mutations in the same row are not necessarily orthologs. Convergent evolution between systems occurred more often than
expected by chance for all functional categories except those labeled ns (not significant) (Table S6 and SI Methods).
Traverse et al. PNAS Early Edition
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repeated sequencing of clones and the evolving community as
a whole, revealed more complex dynamics. Particularly notable
was an example of competition over limiting iron between eco-
types that eventually remodeled the community, but contrary to
expectations new specialist ecotypes actually had evolved re-
currently from the source population of S (Fig. 3). These high-
biofilm, small-colony variants evolved by a variety of single
mutations in relative few pathways (often in wsp). More broadly,
ecotype niche breadth correlated with evolutionary potential,
because generalist S genotypes gave rise to new specialist R and
W types, but the converse did not occur.
The order of adaptive mutations in each lineage also revealed
the prevailing selective forces over time. At the start of the ex-
periment, cells that adhered better to the plastic bead were fa-
vored; consequently, the first three successful ecotypes acquired
mutations in genes (M1, M15, and M16) that are likely re-
sponsible for controlling levels of c-di-GMP, which is known to
underlie transition to a sessile lifestyle (20). These alleles also
altered colony morphology and excluded the ancestor type,
suggesting that variation in c-di-GMP production is sufficient for
adaptive differentiation and the subdivision of biofilm labor,
perhaps by varying the tendency to adhere or disperse.
Next, selection for enhanced metabolic efficiency likely domi-
nated, because mutations occurred that affected central metab-
olism and polysaccharide biosynthesis. Each of these mutations
appeared to expand the potential of the S ecotype (Fig. 4) and
enhanced competition versus earlier mutants (Table 2). These
genotypes also subsequently gave rise to new R mutants with
these mutations, indicating that they were globally beneficial and
not specific to a particular niche. Although these mutations may
simply enhance growth on galactose as a sole exogenous carbon
source, adaptation to planktonic growth in the same medium
occurred by somewhat different mechanisms (Table S5). Thus,
these mutations may be specifically adaptive in biofilms by di-
recting resources toward sessile growth.
Iron Competition Engendered Persistence and Evolvability. As de-
scribed previously, two independent mutations in the promoter
sequence of bacterioferritin led to an ecological revolution that
remodeled the community (Figs. 3 and 4). New mutants of S with
up-regulated bacterioferritin (Fig. S2) likely succeeded in other
niches (Fig. 3C) because of their enhanced competitive ability
for limiting iron, and the mutations enabling invasion were novel
wsp alleles (Fig. 3). It is surprising that W mutants with the al-
ternative bacterioferritin mutation failed to spread beyond this
niche, either by mutations suppressing the wrinkly phenotype or
by alternative gain of function. Only the S lineage, which bal-
ances fitness between planktonic and biofilm conditions and
retains motility (4), was able to spawn new R and W types at least
seven times. This S lineage may be more “evolvable” because of
its larger population size or because of an absence of negative
epistasisbetweenitsexistingmutations andniche-specializing wsp
alleles. Alternatively, the ecological breadth of the S population
may have enabled greater access to new mutations that were
beneficial in a subset of conditions. These alternative explan-
ations are central to the debate surrounding evolvability (34–36)
and may be studied tractably in this model biofilm community.
Evolution During Chronic Infections Is, in Part, a Response to the
Biofilm Lifestyle. Most mutations from published studies of CF
lung isolates do not strictly overlap with the mutations found in
this study, as is to be expected. Bacteria face many different
selective pressures in the CF lung that are absent from our
model, such as high levels of antibiotic use, oxygen deprivation
when suspended in mucus, and immune system evasion (5, 10,
11, 16, 37, 38). Also, in principle, the fact that Burkholderia and
Pseudomonas are in different classes of Proteobacteria should
reduce the frequency of convergence. However, most CF isolates
produce robust biofilms, and many form small-colony variants (5,
7, 9, 38), and, notably, the colony morphologies described in the
literature (38–41) as related to infection outcomes are strikingly
similar to those that evolved in this system. Thus, it is remarkable
that the causes of phenotypic variation during this experimental
biofilm selection and during chronic infections both involved
mutations that affected the TCA cycle, iron metabolism, and
RNA polymerase. For example, mutations in OGDH occurred
frequently in a study of B. dolosa (10) and in this system (Table
4), and this function was up-regulated in two independent studies
of clinical P. aeruginosa (13, 42). This form of altered central
metabolism may be favored by evolution in biofilms. Iron ac-
quisition also is known to be a major selective pressure in the CF
lung (10–12), and it appears to be central to the dynamics of this
system, suggesting that perhaps the biofilm environment rather
than an iron-limited host may favor these changes. Convergent
mutations in subunits of RNA polymerase (RNAP) in clinical
B. dolosa and P. aeruginosa (10–12) and in our study are more
difficult to explain, given their global effects on transcription.
Mutations in major σ factors and polymerase subunits have been
widely reported during long-term selection in chemostats (43)
and occurred in our planktonic control population as well; these
changes may optimize global gene regulation for better growth in
minimal medium (44, 45) that requires more anabolism. RNAP
mutations in isolates from infections have been associated with
selection for antibiotic resistance but alternatively may be
responses to selection for to growth in novel nutrient conditions.
Relatively less convergent evolution was found at loci other
than wsp that affect c-di-GMP metabolism. Despite several re-
lated mutations in infectious P. aeruginosa (11, 12), only one such
mutation was reported for B. dolosa (10). These mutations were
among the first to evolve in our model, so one possibility is that
these mutations became common before clinical sampling.
Starkey et al. (40) reported that laboratory-derived rugose small-
colony variants with increased intracellular levels of c-di-GMP
associated with the ability to persist in CF mouse models. As in
our model, the earliest adaptive steps to form a biofilm may
require altered c-di-GMP levels, but the way each population
accomplishes this task may be variable.
Perhaps the most compelling example of convergent evolution
in this system and the mutations identified in CF lung isolates are
the many mutations in genes involving LPS (Table 4). Altered
LPS production could influence biofilm formation directly be-
cause of potentially increased surface adherence and cell-to-cell
coherence (26, 27). In this system, complementation of one of
the key mutants in this pathway adversely affected biofilm pro-
duction (Fig S1). Rather than selection for immune avoidance,
altered LPS during chronic infections may underlie biofilm ad-
aptation. A subset of the 19 mutations found in the B. dolosa
outbreak restored O-antigen synthesis and may have contributed
to virulence in the lung (10), but others did not, suggesting that
mutations in the same pathway may reflect different selective
pressures. One possibility is that these mutants may beadaptive in
Tangled Bank of Biofilms: Perhaps Predictable Dynamics? What do
we make of this coincidence between the adaptive pathways in
our system and the mutated loci occurring in chronic infections,
and why might prior experimental models (32, 46) have captured
fewer of these mutations? We suggest that both the duration of
the experiment (6 mo or 1,050 generations) and its requirement
to maintain flexible life-history strategies may provide some ex-
planation. Our model required a daily cycle of surface coloni-
zation, biofilm growth, dispersal, and then recolonization, i.e.,
selection for “reversible stickiness.” Under these conditions,
pure biofilm-producing specialists might be disadvantaged in the
long run because of their inability to disperse and recolonize the
next plastic bead. In fact, many early-arising biofilm specialist
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lineages ultimately were displaced by derivatives of the more
flexible S lineage (Fig. 2). Such mutants, successful only in the
short term, may resemble those types found in prior studies (31,
32), but the longer-term cyclical requirement for persistence,
dispersal, and recolonization may better resemble the dynamic
of bacterial populations during chronic infections, because life-
style flexibility will be needed in a lung environment with diverse
conditions (e.g., host immune response, oxygen deprivation, and
continuous biofilm establishment and migration).
Phenotypic diversity is known to be common among CF lung
isolates of B. cepacia, Burkholderia multivorans, and B. cenocepacia,
particularly for biofilm formation and EPS production (5, 13, 47).
Seemingly identical genetic relatives isolated from the same lung at
the same time may display distinct differences in the ability to form
biofilms and may occur within multiple patients (5). Variation
in EPS production was associated with different abilities to
initiate biofilm formation and form mature biofilms (47). One
possible interpretation of these findings is that the Burkholderia
isolates within those patients inhabited distinct niches and
functioned as a community, even among many other bacterial
species in the CF lung (48).
We also explored the high degree of competition both within
niches and among ecotypes in these biofilms and suggest that
such forces are more intense than in well-mixed environments
(49). This interference has the effect of maintaining diversity
over longer time scales and could enhance coevolutionary forces.
Thus, evolutionary dynamics in biofilms can be considered ex-
ceptional, because greater variation is maintained and multiple
adaptive mutations are required for any mutant to exclude others
(Table 2) (23). However, these dynamics also may be expected
and may even become predictable. We offer the hypothesis that
the tangled bank of biofilm adaptation (18), in which adaptive
radiations fuel a pattern of ecological succession, ultimately
drives evolution along proscribed paths. If valid, such a model
offers hope for the development of novel therapeutics that
target these pathways. More worrisome, however, is the evolu-
tion of progressively greater synergy within our model, in which
late-stage–related ecotypes are particularly robust (4). Identi-
fying which community members are most central to biofilm
resilience therefore becomes paramount, with the caution that
the key community member may re-evolve once targeted ther-
apy has ended.
Strains and Growth Conditions. The experimental evolution is described in ref.
4. Briefly, a Burkholderia cenocepacia HI2424 isolate was marked with
a Tn7::lacZ construct to enable blue/white screening (50). A single lac+clone
was inoculated into M9 salts supplemented with 3% (171 mM) galactose
(GMM) containing a polystyrene bead (American Educational Products).
Every 24 h, the bead and any adherent bacteria were transferred to a fresh
tube of GMM containing an oppositely marked (white or black) polystyrene
bead. Cultures were enumerated and characterized by plating on half-con-
centration agar plates that were incubated at 37 °C for 24 h and at room
temperature for 48 h. Cultures for fitness assays were grown under identical
conditions except that strains were grown for 24 h in tryptic soy broth
(Fisher) from frozen stocks and then were transferred to the selection en-
vironment. Biofilm production was quantified using standard methods (3)
with sixfold replication of the sequenced S, R, and W clones isolated from
the end of the experiment. Mixed-population biofilm production was
quantified from an aliquot of the final evolved community. Expected biofilm
production (Fig. 1) was calculated by multiplying biofilm production by the
fraction of the community associated with each ecotype and then summing
these values; contributions of each morph to the observed biofilm produced
are estimates based on the frequency of plated morphotypes.
Metagenomic Sequencing and Analysis. Population samples from 43, 71, and
143 d, reflecting ∼315, 735, and 1,050 generations, were grown in the se-
lective environment for 24 h. [These cell-division estimates differ from those
reported in ref. 4 and reflect more precise measurements of the growth
cycle (SI Methods)]. The bead was vortexed in PBS, and genomic DNA was
extracted using standard methods. Libraries for Illumina sequencing were
prepared following the manufacturer’s procedures and were sequenced by
the Joint Genome Institute (http://jgi.doe.gov) with an Illumina GAII using
paired-end 36-bp reads. The average read depth for each metagenome was
189×, 110×, and 141× for the early (315 generations), intermediate (735
generations), and late (1050 generations) time points, respectively. In addi-
tion, representative single clones of S, R, and W types were isolated at 315
and 1,050 generations and sequenced at >100× coverage (SI Methods).
Subsequently, early and late metagenomes were sequenced from five other
replicate biofilm populations (B2–B6) and from a control, planktonically
evolved population P2 (Table S7, SI Methods, and ref. 4).
The complete approach for identifying mutations and estimating allele
frequencies is described in SI Methods. Mapping of short reads to the B.
cenocepacia HI2424 reference sequence (51) was performed using maq-0.7.1
(52) and later was confirmed using BWA (53). Sequences of single clones
were analyzed as having two haplotypes to flag regions of poor alignment,
and sequences of mixed population metagenomes were analyzed at hap-
lotypes = 20, using clones from these populations as controls for false pos-
itives. The high GC content of this genome produced many initial false
positives and anomalously low coverage in some genome regions. Thus, all
putative mutations were examined manually Table S8 (SI Methods) and most
were confirmed by Sanger sequencing.
Phylogeny and Haplotype Identification. To associate mutations in the met-
agenome with both clones and ecotypes, single colonies were obtained by
growing population samples from 525 and 1,050 generations in the selective
environment by plating as described. Ten clones of the three morphotypes at
each time point were chosen randomly and frozen for reference. DNA from
each clone was isolated, and the loci containing each of the putative
mutations were amplified by PCR and sequenced using standard methods
The estimated frequencies of mutations identified in sequenced clones, in
metagenomes, and by manual screening of variant loci from many clones
were integrated to produce a phylogeny of how population B1 evolved and
diversified. For instance, mutations detected in the metagenome were not
yet associated with a clone or ecotype but enabled estimates of their
changing frequencies over time, which allowed us to determine their likely
order of occurrence. Sequencing multiple isolates at several loci confirmed
this order and also allowed us to assemble haplotypes and to associate
mutations with ecotypes, i.e., the mutations associated with the colony
morphotypes at each stage. This approach also captured multiple putatively
given in SI Methods.
Fitness Assays. After overnight growth from clonal freezer stocks and 24 h of
solitary growth in the selection environment, competitors were removed from
Competitions of evolved isolates versus the ancestor were started by in-
oculating 4 mL of GMM with 100 μL of evolved culture and 900 μL of the
ancestor; this ratio enabled more accurate assays because of the relatively low
fitness of the ancestor. Competitions of earlier S clones versus the M3–M7
haplotype were conducted by using lacZ+and lacZ−competitors (Table S9).
Cultures for competitions were started as described for the ancestral com-
petitions, except that 4 mL of GMM was inoculated with 500 μL of earlier S
isolates and 500 μL of M3–M7. Initial ratios were enumerated, and pop-
ulations were incubated at 37 °C in a New Brunswick TC-7 model roller drum
at 50 rpm. After 24 h, the bead was removed, vortexed in 1 mL of PBS, diluted,
and plated. Competitions between S clones were enumerated on tryptic soy
plates supplemented with X-Gal. Selective values were calculated as:
lnð1 þ sÞ
where t = time (generations), E = frequency of evolved, A = frequency of the
ancestor, and s = selective value. Selection rates in the metagenome were
calculated using the same formula, substituting mutation frequencies at
known times in the numerator. Selection rates of ecotype-specific mutations
were calculated by adjusting their frequency to that of the particular ecotype
(e.g., a W-specific mutation should never exceed 10% of the total community).
Additional methods are described in SI Methods.
ACKNOWLEDGMENTS. We thank Laura Benton, Gabrielle Bergeron, Wendy
Carlson, Zhang Qian, Marcus Dillon, Crystal Ellis, Kenneth Flynn, Megan
McLaughlin, and Christopher Waters for helpful discussions and technical
Traverse et al.PNAS Early Edition
| 9 of 10
support and thank three anonymous reviewers for their comments. This re-
search was supported by National Institutes of Health Grant 1R15AI082528
and National Science Foundation Career Award DEB-0845851. Collaborative
work conducted by the U.S. Department of Energy Joint Genome Institute
was supported by the Office of Science of the US Department of Energy
under Contract DE-AC02-05CH11231.
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