Download Extended PDF

A Self-Produced Trigger for Biofilm
Disassembly that Targets
Exopolysaccharide
Ilana Kolodkin-Gal,1,4 Shugeng Cao,2,4 Liraz Chai,3,4 Thomas Böttcher,2 Roberto Kolter,3 Jon Clardy,2,*
and Richard Losick1,*
1Department
of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
3Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA
4These authors contributed equally to this work
*Correspondence: [email protected] (J.C.), [email protected] (R.L.)
DOI 10.1016/j.cell.2012.02.055
2Department
SUMMARY
Biofilms are structured communities of bacteria that
are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often
have a limited lifespan, disassembling as nutrients
become exhausted and waste products accumulate.
D-amino acids were previously identified as a selfproduced factor that mediates biofilm disassembly
by causing the release of the protein component
of the matrix in Bacillus subtilis. Here we report
that B. subtilis produces an additional biofilmdisassembly factor, norspermidine. Dynamic light
scattering and scanning electron microscopy experiments indicated that norspermidine interacts
directly and specifically with exopolysaccharide.
D-amino acids and norspermidine acted together to
break down existing biofilms and mutants blocked
in the production of both factors formed long-lived
biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B. subtilis,
Escherichia coli, and Staphylococcus aureus.
INTRODUCTION
Many bacteria form complex multicellular communities, known
as biofilms, on surfaces and interfaces (Bryers, 2008; O’Toole
et al., 2000). A hallmark of biofilms is an extracellular matrix typically consisting of protein, exopolysaccharide, and sometimes
DNA, that holds the cells together in the community. Biofilms
are of high significance in agricultural, industrial, environmental,
and clinical settings. For example, the soil bacterium Bacillus
subtilis protects plants from a variety of pathogens by forming
biofilms on the roots (Nagórska et al., 2007). Biofilms are inherently resistant to antimicrobial agents and are at the center of
many persistent and chronic bacterial infections (Costerton
et al., 1999). For example, biofilm formation plays a critical role
in many device-related infections, infective endocarditis, urinary
684 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
tract infections and acute septic arthritis by pathogens such
as Staphylococcus aureus and Staphylococcus epidermidis
(Bryers, 2008; Otto, 2008). Biofilms have natural life cycles;
they form under propitious conditions, and they disassemble
as they age (Karatan and Watnick, 2009; Romero and Kolter,
2011).
Here we focus on the biofilm life cycle of B. subtilis, which
under laboratory conditions forms floating biofilms known as
pellicles at the air-liquid interface of standing cultures (Aguilar
et al., 2007; Branda et al., 2001a; Lopez et al., 2009; Vlamakis
et al., 2008). Cells in the pellicles are held together by an extracellular matrix consisting of exopolysaccharide and amyloidlike fibers largely composed of the protein TasA (Branda et al.,
2006a; Branda et al., 2004; Romero et al., 2010). B. subtilis biofilms have a limited life span, maturing after 3 days of incubation
in biofilm-inducing medium at 22 C but disassembling and
releasing individual planktonic cells by 8 days (Kolodkin-Gal
et al., 2010; Romero et al., 2011).
How do cells in the B. subtilis pellicle escape from the matrix
and return to a planktonic existence during biofilm disassembly?
We previously found that conditioned medium from an 8-day-old
culture contains factors that prevent pellicle formation when
added to a fresh culture. One such factor is a mixture of the
D-amino acids D-Tyr, D-Leu, D-Trp, and D-Met. These amino
acids are incorporated into the cell wall peptidoglycan where
they trigger the release of the amyloid fibers from the cell
(Cava et al., 2011; Kolodkin-Gal et al., 2010; Romero et al.,
2011). Fiber release is mediated via an adaptor protein TapA,
which forms D-amino acid-sensitive foci in the cell wall and is
required for the formation of the fibers and their anchorage to
the cell wall (Romero et al., 2011). D-amino acids were also found
to inhibit biofilm formation by other bacteria, such as S. aureus
and Pseudomonas aeruginosa (Hochbaum et al., 2011; Kolodkin-Gal et al., 2010).
Here we report the discovery of a second biofilm-disassembly
factor present in conditioned medium from aging B. subtilis biofilms. The factor is the polyamine norspermidine, and evidence
indicates that it directly interacts with the exopolysaccharide.
The effect was specific in that other closely related polyamines,
such as spermidine, had little biofilm-inhibiting activity. Mutants
Figure 1. Identification of Norspermidine in Conditioned Medium from B. subtilis and Its Effect on
Pellicle Formation
(A) Biofilm-inhibiting factors in conditioned medium.
B. subtilis strain NCBI3610 was grown at 22 C in 12-well
plates in liquid biofilm-inducing medium for 3 or 8 days.
Conditioned medium (500 ml) from an 8-day-old culture
was concentrated on the C-18 column and eluted stepwise with methanol. Shown is the result of growing cells in
fresh medium to which had been added 20 ml of the 25%,
35%, or 40% methanol eluates.
(B) Norspermidine inhibits biofilm formation. Cells of
NCBI3610 were grown in fresh medium containing PBS
buffer (control), norspermidine (100 mM), morpholine
(100 mM), HPLC-purified fatty acid (100 mM), or spermidine (100 mM). Brighter images of the norspermidinetreated cell revealed cells near the bottom of the well.
(C) Detection of norspermidine. Pellicles were collected
from 3- and 8-day-old cultures (100 ml) of the wild-type
(NCBI3610) and from an 8-day-old culture (100 ml) of a
gabT mutant (IKG623). After mild sonication of the pellicles, cells were separated from extracellular material.
(Other experiments showed that norspermidine is largely
found in pellicles.) Norspermidine in the extracellular
material was derivatized with Fmoc-Cl, and the resulting
Fmoc-norspermidine was detected with an Agilent LC/MS
system. Fmoc-norspermidine was detectable in the old
pellicle from wild-type cells but not in the young or mutant
pellicles. See also Figure S1B.
(D and E) Quanitification of the biofilm-inhibiting activity of
norspermidine and spermidine. Pellicle formation of strain
NCBI3610 was tested in the presence of the indicated
concentrations of norspermidine (D) or spermidine (E). See
also Figure S1A.
blocked in the production of both D-amino acids and norspermidine formed long-lived pellicles, and D-amino acids and norspermidine acted together in breaking down existing, mature
pellicles. Thus, B. subtilis produces factors that act in a complementary manner to free cells from the protein and exopolysaccharide components of the matrix. Finally, we report that
norspermidine was effective in inhibiting biofilm formation by
other bacteria, including S. aureus and Escherichia coli.
RESULTS
Norspermidine Is Self-Produced Biofilm-Inhibiting
Factor
Building on earlier work indicating that aging B. subtilis pellicles
produce two biofilm-inhibiting factors, we applied conditioned
medium to a C-18 Sep-Pak column and collected fractions by
using stepwise elution in 5% percent increments from 5%
to 40% methanol. The 25% and 40% eluates contained
compounds active in inhibiting biofilm formation, whereas the
35% eluate was inert (Figure 1A). As reported previously, the
factor in the 40% eluate was a mixture of D-amino acids (Kolodkin-Gal et al., 2010). To identify the second biofilm-inhibiting
factor, we carried out high-performance liquid chromatography
(HPLC) on the 25% methanol eluate by using a phenyl-hexyl
column. Inhibitory activity was recovered with an elution time of
40 min. Proton nucleic magnetic resonance (NMR) analysis of
the active fraction revealed fatty acids, morpholine, and norspermidine. Further purification with a C-18 HPLC column identified
the inhibitory agent as norspermidine, a finding confirmed with
authentic norspermidine, which inhibited biofilm formation at
25 mM (Figure 1B and Figure S1A, available online). Pure morpholine and fatty acids detected by NMR were inactive (Figure 1B).
Norspermidine’s tendency to form strong complexes with fatty
acids, which explains its elution at 25% methanol from the C-18
Sep-Pak column, complicated its quantification. To circumvent
this problem, we treated conditioned medium with 9-fluorenylmethyloxycarbonyl chloride (Fmoc-Cl), which protects the amino
groups of norspermidine as carbamates and thereby prevents
their interaction with other molecules (Molnár-Perl, 2003).
Fmoc-norspermidine was detected and quantified by liquid
chromotography/mass spectrometry (LC/MS) (Figure S1B).
Using this procedure, we found that norspermidine was present
at a concentration of 50–80 mM in 8-day-old, disassembling
pellicles but at a concentration of less than 1 mM in a 3-day-old
pellicle (Figure 1C).
Finally, the effect of norspermidine was specific in that a
closely related polyamine, spermidine, which differs from norspermidine by the presence of an extra methylene group, was
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. 685
Figure 2. Norspermidine Acts Together with
D-amino Acids
(A) Pellicle longevity. Shown are 7-day-old cultures of the
wild-type (WT), a mutant (ΔgbaT) blocked in norspermidine production (IKG623), a double mutant (ΔylmE ΔracX)
blocked in D-amino acid production (IKG55), and a triple
mutant (ΔgbaT ΔylmE ΔracX) blocked in the production of
both (IKG625). See also Figure S2A.
(B) Preventing biofilm formation. Cells were grown for
3 days in medium containing as indicated D-tyrosine
(D-Tyr), norspermidine, a mixture of D-tyrosine, D-methionine, D-leucine, and D-tryptophan (D-aa) and the indicated combinations of amino acids and norspermidine at
the indicated concentrations.
(C) Quantifying pellicle breakdown. On the surface of
3-day-old pellicles were placed droplets (50 ml) containing
buffer (PBS), a mixture of D-tyrosine, D-methionine,
D-leucine, and D-tryptophan each at final concentration of
12.5 mM, norspermidine at a final concentration of 50 mM,
or, as in (C), a combination of D-amino acids each at
a concentration of only 2.5 mM and norspermidine at a
concentration of 10 mM. After incubation for the indicated
times, pellicle material and the medium were separated
and each brought to a volume of 3 ml. After mild sonication, the OD600 was determined for each sample. The
percentage of disassembly represents the OD600 of
the medium as a percent of the sum of the OD600 of the
medium and the OD600 of the pellicle. The results are
an average of three independent experiments done in
duplicate. The error bars indicate standard deviation.
inactive in inhibiting biofilm formation at concentrations up to
2 mM (Figures 1D and 1E).
A Mutant Blocked in Both D-Amino Acid and
Norspermidine Production Forms Long-Lived Biofilms
To evaluate the contribution of norspermidine to biofilm
disassembly genetically, we created a mutation blocking its
production. Norspermidine is synthesized from aspartateb-semialdehyde in a pathway involving the enzyme L-diaminobutyric acid transaminase (Lee et al., 2009). We constructed
a mutant lacking the B. subtilis gene (gabT) encoding this
enzyme and found that the mutant was blocked in norspermidine
production (Figure 1C) and was partially impaired in biofilm
disassembly (Figure 2A). The gabT mutant formed pellicles that
remained relatively thick at a time (day 7) when the wild-type
had undergone substantial disassembly. Nonetheless, the
mutant pellicle had lost the wrinkly phenotype characteristic of
young biofilms by day 7. We wondered whether the contribution
of norspermidine to biofilm disassembly might be partially
redundant with that of D-amino acids, which are produced by
racemases encoded by racX and ylmE. Like a gabT mutant,
a racX ylmE double mutant was partially impaired in biofilm
disassembly. Strikingly, however, a gabT racX ylmE triple mutant
formed robust pellicles that retained their wrinkly phenotype at
a time (7 days) when the wild-type had substantially disassembled (Figure 2A). As a further test of the involvement of
norspermidine in biofilm disassembly, we constructed a mutant
lacking carboxynorspermidine decarboxylase, which catalyzes
the last step in the biosynthetic pathway. As in the case of the
gabT mutant, a mutant lacking the B. subtilis homolog (yaaO)
686 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
of the decarboxylase gene was partially impaired in biofilm
disassembly, and a yaaO racX ylmE triple mutant formed
pellicles that remained intact at a time when the wild-type had
disassembled (Figure S2A).
D-Amino Acids and Norspermidine Act Together in
Preventing Biofilm Formation and Triggering Biofilm
Disassembly
These findings suggest that norspermidine and D-amino acids
act by different mechanisms to trigger biofilm disassembly.
Consistent with this idea, combinations of norspermidine with
D-Tyr or with a mixture of D-Met, D-Trp, D-Leu, and D-Tyr effectively prevented biofilm formation at concentrations that were
ineffective in blocking biofilm formation when applied separately
(Figure 2B). Similarly, a mixture of D-amino acids and norspermidine was more effective in causing the breakdown of an existing
biofilm than were either D-amino acids or norspermidine alone
(Figure 2C). Thus, D-amino acids and norspermidine act together
in preventing biofilm formation and triggering the disassembly of
mature biofilms.
Norspermidine Targets the Exopolysaccharide
How does norspermidine trigger biofilm disassembly? A clue
came from the observation that the residual pellicle (wispy fragments of floating material with some structure) produced in the
presence of norspermidine resembled pellicles seen for a mutant
blocked in exopolysaccharide production but not those (thin,
featureless pellicles) seen for a mutant blocked in amyloid-fiber
production. More importantly, norspermidine had little effect on
the residual pellicle produced by an exopolysaccharide mutant
Figure 3. Norspermidine Disrupts Exopolysaccharide
Shown are phase contrast and fluorescence images of
cells of the wild-type (WT; NCBI3610) harvested from
pellicles grown in the presence or absence (untreated)
of norspermidine (25 mM) or a high concentration of
spermidine (1 mM). The cells were washed in PBS and
stained for exopolysaccharide with a conjugate of
concanavalin A with Texas red. See also Figure S2B and
Figure S3.
but abolished pellicle formation by the amyloid-fiber mutant.
In other words, the effect of norspermidine was synergistic
with that of a mutation blocking fiber formation but not with
a mutant blocked in exopolysaccharide production (Figure S2B).
These observations suggested that norspermidine was interfering with the exopolysaccharide component of the matrix.
To investigate this hypothesis, we visualized exopolysaccharide by fluorescence microscopy by using a conjugate of the
carbohydrate-binding protein concanavalin A with Texas red
(Figure 3) (McSwain et al., 2005). As evidence of specificity, the
conjugate decorated wild-type cells but not cells from a mutant
(Δeps) blocked in exopolysaccharide production (Figure S3A).
Indeed, at an exposure at which concanavalin A staining with
the wild-type strain gave an extremely bright fluorescent signal,
little or no signal could be detected for the Δeps mutant, except
at long exposures and enhanced brightness (Figure S3A). Next,
we investigated the effects of norspermidine and spermidine.
Figure 3 shows that norspermidine treatment disrupted the
relatively uniform, cell-associated pattern of staining seen with
untreated cells, resulting in isolated patches of fluorescence.
No such effect was seen with cells treated with spermidine.
Simply mixing norspermidine with concanavalin A did not
quench the intensity of fluorescence of the flurophore (data not
shown). Hence, the difference in the staining was evidently due
to differential levels of cell-associated exopolysaccharide. As
a control, and in contrast to the results seen with concanavalin
A, norspermidine had little or no effect on the protein component
of the matrix as judged with a functional fusion of TasA to the
fluorescent protein mCherry (Figure S3B) (Kolodkin-Gal et al.,
2010). We infer that norspermidine disrupts the matrix and
apparently does so by targeting exopolysaccharide.
Norspermidine but Not Spermidine Interacts
with Exopolysaccharide
The expression of the operons epsA-O and yqxM-sipW-tasA,
which specify the exopolysaccharide and protein components
of the extracellular matrix, respectively, was
not measurably impaired by the addition of
norspermidine (Figure S4). Also, cell growth
was not significantly inhibited by norspermidine (Figure S4). We therefore considered the
possibility that norspermidine was interacting
with the exopolysaccharide directly. To attempt
to detect such an interaction, we used dynamic
light scattering, a standard procedure for
measuring the average radius of polymers
(Berne and Pecora, 1976; Orgad et al., 2011; Vinayahan et al.,
2010).
Figure 4A shows that purified exopolysaccharide exhibited
an average radius of 585 ± 40 nm at pH 5.5, presumably representing an effective radius for the interacting polymers. Strikingly, treatment of the exopolysaccharide with norspermidine
reduced the average radius substantially (175 ± 10 nm), whereas
treatment with spermidine had only a small effect on the average
radius (500 ± 20 nm). This indicates that the specificity of norspermidine resulted from a direct interaction with exopolysaccharide. The effect of norspermidine was seen over a range of
exopolysaccharide concentrations (1–30 mg/ml) (Figure 4A and
data not shown) and also at pH 7 (Figure 4A).
As an independent approach to detecting an interaction
between norspermidine and exopolysaccharide, we carried out
scanning electron microscopy. Purified exopolysaccharide was
seen to be in the form of aggregates, which had an average
diameter of 570 nm (Figure 4B; data not shown). Strikingly,
the addition of norspermidine reduced the diameter of the
aggregates to 85 nm (Figure 4B; data not shown). Once again,
and, as a demonstration of specificity, spermidine had little
effect on the size of the aggregates.
Use of a Panel of Small Molecules Reveals Features
Important for Biofilm-Inhibitory Activity
To identify features of norspermidine important for its biofilmdisassembly activity, we tested a library of polyamines in our
biofilm inhibition assay (see Figures 5A and 5B, Figure S5, and
Table S2). In addition to norspermidine (1), we found that
norspermine (2) exhibited biofilm-inhibitory activity against
B. subtilis. These molecules have in common a motif consisting
of three methylene groups flanked by two amino groups. The
motif is present twice in (1) and three times in (2). Another polyamine, 1,3-diaminopropane (6), has only one copy of the motif
and was significantly less active (>5 mM). Also relatively inactive
(inhibition was only observed at concentrations above 2 mM)
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. 687
Figure 4. Norspermidine Interacts with Exopolysaccharide Polymers
(A) Dynamic light scattering. Listed are the average
hydrodynamic radii of the exopolysaccharide as measured by dynamic light scattering. Exopolysaccharide was
purified from pellicles. Light scattering was measured for
exopolysaccharide alone as well as for exopolysaccharide
that had been mixed with 0.75 mM norspermidine or with
0.75 mM spermidine. Shown are the results obtained in the
absence of polyamine (black) in the presence of norspermidine (white) and in the presence of spermidine
(gray) with exopolysaccharide at the indicated concentrations and pH. Error bars represent the standard deviation of polymer radii in a single sample.
(B) Scanning electron microscopy Purified exopolysaccharide was dissolved in DDW at a final concentration
of 10 mg/ml and mixed with either norspermidine or
spermidine (0.75 mM final concentration). Samples were
prepared as described in Experimental Procedures.
Shown are three different magnifications of representative
fields showing exopolysaccharide alone (EPS) and exopolysaccharide that had been mixed with norspermidine
(EPS + norspermidine) or with spermidine (EPS + spermidine). See Figure S4 for controls showing little effect on
growth or eps transcription.
were spermidine (3), spermine (4), and putrescine (5), which
each have a pair of amines separated by four methylenes, and
cadaverine (19), which has a pair of amines separated by five
methylenes. Replacing some or all of the amines in norspermidine with tertiary amines (7–9, 18), replacing the secondary
amine with an ether linkage (10) or eliminating two or all of the
amines (20, 11) resulted in molecules that were relatively inactive. Whereas replacing the terminal amines with tertiary amines
resulted in inactivity, in one case (21) the presence of a tertiary
amine at the middle position did not block activity. Importantly,
the charge of each amine (at the neutral pH of the medium)
was also important for biofilm-inhibiting activity. Molecules that
had neutral amide bonds instead of amines separated by three
methylenes (15–17) were only weakly active or inactive (Figure S5
and Table S2).
We conclude that the structure and the charge of the polyamine are required for biofilm-inhibiting activity. In particular,
a motif consisting of three methylene groups flanked by two
positively charged amino groups is favored for high biofilm-inhibiting activity. Reinforcing this hypothesis, three additional,
synthetic polyamines exhibiting this motif (12, 13 and 14) were
active (Figure S5).
Norspermidine but Not Spermidine Inhibits Biofilm
Formation by S. aureus and E. coli
We wondered whether polyamines might prevent biofilm formation by other bacteria that produce an exopolysaccharide matrix.
Indeed, the same molecules that inhibited biofilm formation by
688 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
B. subtilis were effective in inhibiting biofilm
formation (but not growth; data not shown)
by S. aureus and E. coli, whereas those that
were inactive with B. subtilis were not (Figures
6 and 7 and Figure S6). In testing E. coli we chose the biofilmproficient strain MC4100 because a major component of exopolysaccharide is colanic acid (Danese et al., 2000; Price and
Raivio, 2009). Colanic acid is a negatively charged polymer,
and light scattering experiments indicated a direct interaction
with norspermidine (data not shown). Reinforcing the idea that
norspermidine was targeting the exopolysaccharide, fluorescence microscopy experiments analogous to those presented
above for B. subtilis showed markedly diminished staining of
exopolysaccharide when cells of S. aureus and E. coli were
treated with norspermidine but not spermidine (data not shown).
Interestingly, and in contrast to the above results, norspermidine is reported to promote rather than inhibit biofilm formation
by Vibrio cholerae (Lee et al., 2009). Evidence indicates that in
V. cholerae norspermidine acts to de-repress biofilm formation
via a signal transduction mechanism (Karatan et al., 2005). Nonetheless, and despite the V. cholerae exception, our results
support the concept that biofilm formation can be prevented
and existing biofilms disrupted by molecules that interact
directly with exopolysaccharides.
DISCUSSION
B. subtilis forms architecturally complex biofilms (pellicles) at the
air/liquid interface of standing cultures (Aguilar et al., 2007;
Branda et al., 2001b). These floating communities are transient;
they mature after 3 days in biofilm-inducing medium but then
disassemble by 8 days, releasing individual planktonic bacteria
Figure 5. Structure Activity Relationship Study of Norspermidine
(A) Shows compounds tested for biofilm-inhibiting activity.
(B) Shows the effect of the numbered compounds on pellicle formation by B. subtilis (NCBI3610). The compounds were tested at 200 mM.
(C and D) show the results of modeling the interaction of norspermidine and spermidine with an acidic exopolysaccharide. Norspermidine binds via salt
bridges between amino and carboxyl groups (dotted lines) in a clamp-like mode across the exopolysaccharide secondary structure of a disaccharide repeat.
[a(1,6)Glc-b(1,3)GlcA]n. Whereas norspermidine aligns well with the repeat, the spacing of amino groups of spermidine does not match the symmetric pattern
of anionic side groups, implying weaker affinity. See supporting information for modeling of plausible interactions with other charged and non-charged
polysaccharide structures. For tests of additional molecules see Figure S5 and Table S2.
(Kolodkin-Gal et al., 2010). Cells in the biofilm are held together
by exopolysaccharide and amyloid-like fibers largely consisting
of TasA (Branda et al., 2006b; Branda et al., 2004; Romero et al.,
2010). The return of cells in the biofilm to a planktonic state must
therefore involve mechanisms for their release from the exopolysaccharide and protein components of the matrix. One such
mechanism is the production late in the life cycle of the D-amino
acids D-Tyr, D-Leu, D-Trp, and D-Met, which are incorporated
into the peptidoglycan where they trigger the release of the
TasA fibers (Kolodkin-Gal et al., 2010). This release is mediated
by an adaptor protein, TapA, which forms D-amino acid-sensitive foci in the cell wall (Romero et al., 2011). Here we have
reported the discovery of a second biofilm-disassembly factor,
norspermidine, which is also produced late in the life cycle
of the biofilm and is required for complete disassembly of
B. subtilis biofilms. Importantly, mutants blocked in the production of both D-amino acids and norspermidine formed long-lived
pellicles that retained their architectural complexity for extended
periods of time. We do not fully know the mechanism(s) by which
the production of norspermidine and D-amino acids is delayed
until late in the biofilm life cycle but experiments based on the
use of lacZ fused to genes involved in norspermidine (gabT
and yaaO) and D-amino acid biosynthesis (the racemase genes
racX and ylmE) indicate that regulation occurs at the level of
gene transcription (L. Silverstein, Y. Chai, I.K.-G., unpublished
results).
The biofilm-inhibiting effect of norspermidine was specific in
that a closely related polyamine, spermidine (differing only by
an extra methylene group), exhibited little activity. Interestingly,
another polyamine, norspermine, was also active in biofilm inhibition, whereas its close relative spermine (once again having an
extra methylene) was inactive. These results and the results of
using a panel of seventeen additional compounds suggest that biofilm inhibition depends on a motif of two or three pairs of primary
or secondary charged amines separated by three methylenes.
Several lines of evidence indicate that norspermidine acts in
a complementary manner to D-amino acids by targeting the
exopolysaccharide. First, norspermidine and D-amino acids
acted cooperatively in inhibiting biofilm formation, suggesting
that they function by different mechanisms. Second, pellicles
formed in the presence of norspermidine resembled the wispy,
fragmented material produced by an exopolysaccharide mutant
but not the thin, flat, featureless pellicle of a mutant blocked
in amyloid-fiber production. Third, fluorescence microscopy
showed that norspermidine (but not spermidine) disrupted the
normal uniform pattern of staining of exopolysaccharide but
had little effect on the staining pattern of the protein component
of the matrix. Finally, and most directly, light scattering and
electron microscopy experiments revealed that norspermidine,
but not spermidine, interacted with purified exopolysaccharide.
Remarkably, the biofilm-inhibiting effect of norspermidine and
norspermine was not limited to B. subtilis. Both molecules
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. 689
Figure 6. Norspermidine Inhibits Biofilm Formation by S. aureus
(A) The effect of the numbered compounds displayed in
Figure 5A on the formation of submerged biofilms by
S. aureus strain SCO1. The compounds were tested at
500 mM. Biofilm formation was visualized by CV staining of
submerged biofilms.
(B) Quantification of the effects of norspermidine, norspermine, spermine, and spermidine as measured by CV
staining (see Experimental Procedures). The results are an
average of three independent experiments done in triplicate. The error bars indicate standard deviation. See also
Figure S6.
inhibited the formation of submerged biofilms by S. aureus and
E. coli. Indeed, the same pattern of molecules that were active
or inactive in inhibiting biofilm formation by B. subtilis was
observed for S. aureus and E. coli. It is therefore attractive to
posit that the broad spectrum of norspermidine and norspermine
reflects a common mechanism of targeting the exopolysaccharide. Indeed, this was supported by fluorescence microscopy
with S. aureus and E. coli and light scattering experiments with
purified exopolysaccaride from E. coli.
However, we do not exclude the possibility
that norspermidine also targets DNA, which is,
of course, negatively charged and is known to
be a component of the matrix for certain
bacteria, such as S. aureus (Branda et al., 2005).
What is the nature of interaction of norspermidine with exopolysaccharides? Exopolysaccharides often contain negatively charged
residues (e.g., uronic acid) or neutral sugars
with polar groups (e.g., poly-N-acetylglucosamine) (Kropec
et al., 2005; Sutherland, 2001). Molecular modeling suggests
that the amines in norspermidine, but not those in spermidine,
are capable of interacting with such charged (Figures 5C and
5D) or polar groups (Figure S7) in secondary structure of the
exopolysaccharide. We suggest that this interaction enhances
the ability of the polymers to interact with each other or with other
parts of the polymer chain. Indeed, the results of fluorescence
Figure 7. Norspermidine Inhibits Biofilm Formation by E. coli
(A) The effect of the numbered compounds shown in
Figure 5A on submerged biofilm formation by E. coli strain
MC4100. The compounds were tested at 500 mM. Biofilm
formation was visualized by CV staining of submerged
biofilms.
(B) shows quantification of the effects of norspermidine,
norspermine, spermine, and spermidine as measured by
CV staining (see Experimental Procedures). The results
are an average of three independent experiments done in
triplicate. The error bars indicate standard deviation. See
also Figure S6.
690 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
microscopy (Figure 3), dynamic light scattering (Figure 4A), and
scanning electron microscopy (Figure 4B) appear to indicate
that the exopolysaccharide network collapses upon addition of
norspermidine. We speculate that exopolysaccharide polymers
form an interwoven meshwork in the matrix that helps hold cells
together and that condensation of the polymers in response to
norspermidine weakens the meshwork and causes release of
polymers.
Given the apparent versatility of norspermidine and norspermine in inhibiting biofilm formation by a variety of bacteria, it is
conceivable that these and other, tailor-made polyamines that
bind with high affinity to specific exopolysaccharides might
offer a general approach (in conjunction with D-amino acids)
to preventing biofilm formation by medically and industrially
important microorganisms. Indeed, in preliminary experiments
we have succeeded in synthesizing polyamine-like molecules
with enhanced potency in blocking biofilm formation by
S. aureus that were designed on the basis of model building
for optimal interaction with poly N-acetyl glucosamine (data
not shown).
EXPERIMENTAL PROCEDURES
Colony and Pellicle Formation
For pellicle formation in liquid medium, cells were grown to exponential phase,
and 3 ml of culture were mixed with 3 ml of medium in a 12-well plate (VWR).
Plates were incubated at 23 C for 3 days. Images of the pellicles were
recorded similarly.
Submerged Biofilm Formation
For S. aureus, cells were grown in Luria-Bertani (LB) overnight and then diluted
1:1,000 in Tryptic Soy Broth (Sigma), applied with 3% NaCl and 0.5% glucose.
Plates were incubated at 37 C for 24 hr. For E. coli, cells were grown in LB
overnight, then diluted 1:100 in M9 and applied with 0.02% Casamino acid
solution and 0.5% glycerol. Plates were incubated in 30 C for 3 days.
Preparing Conditioned Medium
B. subtilis 3610 or its derivatives were grown in LB medium to exponential
phase. A total of 1 ml of culture was then applied to 100 ml of MSgg medium
and grown in a 500 liter flask at 22 C. Next, pellicles and conditioned medium
were collected by centrifugation at 8,000 rpm for 15 min. The conditioned
medium (supernatant fluid) was removed and filtered through a 0.22 mm filter.
The filtrates were stored at 4 C. For further purification the biofilm-inhibiting
material was fractionated on a C-18 Sep Pak cartridge with a stepwise elution
of 0% to 60% methanol with steps of 5%.
Separation, Identification, and Quantification of Norspermidine
See Supplemental Information for details.
Fluorescence Microscopy
Fluorescence microscopy was carried out with 3-day-old pellicles. For
TasA-mcherry detection, cells were washed with PBS buffer and suspended
in 50 ml of PBS buffer. For exopolysaccharide detection, pellicles were
collected and washed with PBS. The cells were labeled by replacing the
PBS with Texas red-concanavalin A (50 mM) and incubating with shaking in
room temperature for an hour. The cells were rinsed again with PBS, placed
on poly-L-lysine (Sigma) pretreated slides and then imaged with an Olympus
workstation. Cells were imaged with various exposure times and images
were taken under conditions in which concanavalin A staining was largely
specific to exopolysaccharide (see Figure S3). Images were taken with an
automated software program, SimplePCI.
Crystal Violet Staining
Crystal Violet (CV) staining was done as described previously except that the
cells were grown in 12-well plates (O’Toole and Kolter, 1998). Wells were
stained with 500 ml of 1.0% CV dye, rinsed twice with 2 ml doubly distilled
water (DDW), and thoroughly dried. For quantification, 1 ml of 95% ethanol
was added to each well. Plates were incubated for 1 hr at room temperature
with shaking. CV solution was diluted and the optical density (OD) at 595 nm
was measured with Ultraspec 2000 (Pharmacia Biotech).
Exopolysaccharide Purification
Pellicles were harvested in day 3, washed twice in PBS, and mildly sonicated.
Cells were removed by centrifugation. The supernatant fluid was mixed with
cold isopropanol in a 5:1 ratio and incubated at 4 C overnight. Samples
were centrifuged at 8,000 rpm, 4 C, for 10 min. Pellets were resuspended
in a digestion mix of 0.1 M MgCl2, 0.1 mg/ml DNase, and 0.1 mg/ml RNase
solution, mildly sonicated, and incubated for 4 hr at 37 C. Samples were
extracted twice with phenol-chloroform. The aquatic fraction was dialyzed
for 48 hr with Slide-A-Lyzer Dialysis cassettes by Thermo Fisher, 3,500
MCWO. Samples were lyophilized.
Dynamic Light Scattering
See Supplemental Information for details.
Scanning Electron Microscopy
Exopolysaccharide was dissolved in DDW at a final concentration of 10 mg/ml
and mixed with norspermidine or spermidine (0.75 mM final concentration).
Samples were then spotted onto poly-L-lysine-coated Si surfaces and kept
in a humid environment for 30 min. The Si pieces were then rinsed with
DDW. To capture the native, hydrated, state of exopolysaccharide, we critical-point dried the samples. Images were obtained with a Zeiss Supra55 Field
Emission scanning electron microscope. For further details see Extended
Experimental Procedures.
Primers
See Table S1.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and two table and can be found with this article online at
doi:10.1016/j.cell.2012.02.055.
ACKNOWLEDGMENTS
We thank T. Kodger for help with dynamic light scattering, C. Marks and
A. Grahame for their help with sample preparation for electron microscopy
imaging, and the Harvard Center for Nanoscale Systems for use of its Imaging
Facility. I.K.-G. is a fellow of the Human Fronteir Science Program. T.B. was
supported by German Academy of Sciences Leopoldina (LPDS 2009-45),
Leopoldina Research Fellowship. This work was supported by National
Institutes of Health grants GM18568 to R.L., GM58213 and GM82137 to
R.K., GM086258 and AI057159 to J.C. and by the BASF Advanced Research
Initiative at Harvard University to R.L. and R.K. Harvard University has filed
a patent application based on this work.
Received: September 11, 2011
Revised: December 22, 2011
Accepted: February 8, 2012
Published: April 26, 2012
REFERENCES
Aguilar, C., Vlamakis, H., Losick, R., and Kolter, R. (2007). Thinking about
Bacillus subtilis as a multicellular organism. Curr. Opin. Microbiol. 10, 638–643.
Berne, B.J., and Pecora, R. (1976). Dynamic Light Scattering (New York: Wiley).
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. 691
Branda, S.S., González-Pastor, J.E., Ben-Yehuda, S., Losick, R., and Kolter,
R. (2001a). Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci.
USA 98, 11621–11626.
Branda, S.S., González-Pastor, J.E., Ben-Yehuda, S., Losick, R., and Kolter,
R. (2001b). Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci.
USA 98, 11621–11626.
Branda, S.S., González-Pastor, J.E., Dervyn, E., Ehrlich, S.D., Losick, R., and
Kolter, R. (2004). Genes involved in formation of structured multicellular
communities by Bacillus subtilis. J. Bacteriol. 186, 3970–3979.
Branda, S.S., Vik, S., Friedman, L., and Kolter, R. (2005). Biofilms: the matrix
revisited. Trends Microbiol. 13, 20–26.
widespread in bacteria and essential for biofilm formation in Vibrio cholerae.
J. Biol. Chem. 284, 9899–9907.
Lopez, D., Vlamakis, H., and Kolter, R. (2009). Generation of multiple cell types
in Bacillus subtilis. FEMS Microbiol. Rev. 33, 152–163.
McSwain, B.S., Irvine, R.L., Hausner, M., and Wilderer, P.A. (2005). Composition and distribution of extracellular polymeric substances in aerobic flocs and
granular sludge. Appl. Environ. Microbiol. 71, 1051–1057.
Molnár-Perl, I. (2003). Quantitation of amino acids and amines in the same
matrix by high-performance liquid chromatography, either simultaneously or
separately. J. Chromatogr. A 987, 291–309.
Branda, S.S., Chu, F., Kearns, D.B., Losick, R., and Kolter, R. (2006a). A major
protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 59,
1229–1238.
Nagórska, K., Bikowski, M., and Obuchowski, M. (2007). Multicellular
behaviour and production of a wide variety of toxic substances support
usage of Bacillus subtilis as a powerful biocontrol agent. Acta Biochim. Pol.
54, 495–508.
Branda, S.S., Chu, F., Kearns, D.B., Losick, R., and Kolter, R. (2006b). A major
protein component of the Bacillus subtilis biofilm matrix. Mol. Microbiol. 59,
1229–1238.
O’Toole, G.A., and Kolter, R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30,
295–304.
Bryers, J.D. (2008). Medical biofilms. Biotechnol. Bioeng. 100, 1–18.
Cava, F., de Pedro, M.A., Lam, H., Davis, B.M., and Waldor, M.K. (2011).
Distinct pathways for modification of the bacterial cell wall by non-canonical
D-amino acids. EMBO J. 30, 3442–3453.
Costerton, J.W., Stewart, P.S., and Greenberg, E.P. (1999). Bacterial biofilms:
a common cause of persistent infections. Science 284, 1318–1322.
Danese, P.N., Pratt, L.A., and Kolter, R. (2000). Exopolysaccharide production
is required for development of Escherichia coli K-12 biofilm architecture.
J. Bacteriol. 182, 3593–3596.
O’Toole, G., Kaplan, H.B., and Kolter, R. (2000). Biofilm formation as microbial
development. Annu. Rev. Microbiol. 54, 49–79.
Orgad, O., Oren, Y., Walker, S.L., and Herzberg, M. (2011). The role of alginate
in Pseudomonas aeruginosa EPS adherence, viscoelastic properties and cell
attachment. Biofouling 27, 787–798.
Otto, M. (2008). Staphylococcal biofilms. Curr. Top. Microbiol. Immunol. 322,
207–228.
Price, N.L., and Raivio, T.L. (2009). Characterization of the Cpx regulon in
Escherichia coli strain MC4100. J. Bacteriol. 191, 1798–1815.
Hochbaum, A.I., Kolodkin-Gal, I., Foulston, L., Kolter, R., Aizenberg, J., and
Losick, R. (2011). Inhibitory effects of D-amino acids on Staphylococcus
aureus biofilm development. J. Bacteriol. 193, 5616–5622.
Romero, D., and Kolter, R. (2011). Will biofilm disassembly agents make it to
market? Trends Microbiol. 19, 304–306.
Karatan, E., and Watnick, P. (2009). Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol. Mol. Biol. Rev. 73,
310–347.
Romero, D., Aguilar, C., Losick, R., and Kolter, R. (2010). Amyloid fibers
provide structural integrity to Bacillus subtilis biofilms. Proc. Natl. Acad. Sci.
USA 107, 2230–2234.
Karatan, E., Duncan, T.R., and Watnick, P.I. (2005). NspS, a predicted
polyamine sensor, mediates activation of Vibrio cholerae biofilm formation
by norspermidine. J. Bacteriol. 187, 7434–7443.
Romero, D., Vlamakis, H., Losick, R., and Kolter, R. (2011). An accessory
protein required for anchoring and assembly of amyloid fibres in B. subtilis
biofilms. Mol. Microbiol. 80, 1155–1168.
Kolodkin-Gal, I., Romero, D., Cao, S., Clardy, J., Kolter, R., and Losick, R.
(2010). D-amino acids trigger biofilm disassembly. Science 328, 627–629.
Sutherland, I. (2001). Biofilm exopolysaccharides: a strong and sticky framework. Microbiology 147, 3–9.
Kropec, A., Maira-Litran, T., Jefferson, K.K., Grout, M., Cramton, S.E., Götz, F.,
Goldmann, D.A., and Pier, G.B. (2005). Poly-N-acetylglucosamine production
in Staphylococcus aureus is essential for virulence in murine models of
systemic infection. Infect. Immun. 73, 6868–6876.
Vinayahan, T., Williams, P.A., and Phillips, G.O. (2010). Electrostatic interaction and complex formation between gum arabic and bovine serum albumin.
Biomacromolecules 11, 3367–3374.
Lee, J., Sperandio, V., Frantz, D.E., Longgood, J., Camilli, A., Phillips, M.A.,
and Michael, A.J. (2009). An alternative polyamine biosynthetic pathway is
692 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
Vlamakis, H., Aguilar, C., Losick, R., and Kolter, R. (2008). Control of cell fate by
the formation of an architecturally complex bacterial community. Genes Dev.
22, 945–953.
Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Strains and Media
Bacillus subtilis strains PY79, 3610 and their derivatives were grown in LB medium at 37 C or MSgg medium (Branda et al., 2001) at
23 C. Solid media contained 1.5% Bacto agar. When appropriate, antibiotics were added at the following concentrations for growth
of B. subtilis: 10 mg per ml of tetracycline, and 5 mg per ml of erythromycin, 500 mg per ml of spectinomycin.
Strains used in this work:
PY79, a derivative of B. subtilis 168, was used as a host for transformation.
3610, a wild strain of B. subtilis (NCBI 3610), which is capable of forming robust biofilms (Branda et al., 2001).
Staphylococcus aureus SC01was obtained from the Kolter lab collection (López and Kolter, 2010).
E. coli strain MC4100 was obtained from the Kolter lab collection.
Pseudomonas aeruginosa PA14 was obtained from the Kolter lab collection.
Strain DS76, 3610 Deps::tet (lab stock).
Strain FC55, 3610 DtasA::spec (lab stock).
Strain FC5, 3610 containing PepsA-lacZ at the amyE locus and a cat gene.
Strain IKG55, 3610 containing DracX::spec and DylmE::tet (Kolodkin-Gal et al., 2010).
Strain DR30, 3610 containing tasA-mCherry at the amyE locus and a cat gene.
Strain IKG624, 3610 containing DyaaO::tet (this work).
Strain IKG623, 3610 containing DgabT::spec (this work).
Strain IKG625, 3610 containing DracX::mls and DylmE::tet, DgbaT::kan (this work).
Strain IKG626, 3610 containing DracX::spec and DylmE::mls, DyaaO::tet (this work).
Strain Construction
Strains were constructed via standard methods (Sambrook and Russell, 2001). Long-flanking PCR mutagenesis was used to
create DgbaT::spec and DyaaO (Wach, 1996). Primers are described in the table below. DNA was introduced into lab strains by
DNA-mediated transformation of competent cells (Gryczan et al., 1978). SPP1 phage-mediated transduction was used to move
antibiotic resistance marker-linked mutations from lab strains to the wild strain 3610 (Yasbin and Young, 1974).
Reagents
Norspermidine (1), norspermine (2), spermidine (3), spermine (4), putrescine (5), 1,3-diaminopropane (6), 1,1,5,9,9-N,N,N,N,
(7), 1,1,9,9-N,N,N,
N-pentamethylnorspermidine
[N1-(3-(dimethylamino)propyl)- N1,N3,N3-trimethylpropane-1,3-diamine
N-tetramethylnorspermidine [N1-(3-(dimethylamino)propyl)-N3,N3-dimethylpropane-1,3-diamine (8), 5-N-methylnorspermidine
[(3-aminopropyl)- N1-methylpropane-1,3-diamine (9), 3,30 -oxybis(propan-1-amine) (10), 3,7,11,18,22,26-Hexaazatricyclo
[26.2.2.213,16]tetratriaconta-13,15,28,30,31, 33-hexaene (12), N1-dodecyl-N3-(3-(dodecylamino)propyl)propane-1,3-diamine (13),
di-tert-butyl 5,50 -((3,4-dimethyl-1H-pyrrole-2,5-diyl)bis(methylene))bis(3,4-dimethyl-1H-pyrrole-2-carboxylate) (14), 2,20 -(1H-pyrrole2,5-diyl)di(acetohydrazide) (15), 3,30 -(2,2-dimethylhydrazine-1,1-diyl)dipropanamide (16), N,N’-(azanediylbis(propane-3,1-diyl))
bis(2,3,4,5,6-pentahydroxyhexanamide) (18), pentane-1,5-diamine (19), 3,30 -azanediylbis(propan-1-ol) (20), and N1,N1-bis(3-aminopropyl)propane-1,3-diamine (21) were obtained from Sigma-Aldrich (Atlanta, GA). N,N’-(azanediylbis(propane-3,1-diyl))bis(2,3,4,5,6pentahydroxyhexanamide) (17) was purchased from Toronto research chemicals (Toronto, Canada) (O’Toole and Kolter, 1998). Diethyl 4-oxoheptanedioate (O’Toole and Kolter, 1998) was synthesized according to the literature from furylacrylic acid (Emerson and
Longley, 1963). The product was purified by distillation as described previously and identified by 1H NMR and 13C NMR. Texas redconcanavalin A was obtained from Invitrogen-Molecular Probes (Eugene, OR).
Separation and Identification of Norspermidine
The biofilm-inhibiting, 25% methanol eluate of the conditioned medium from an 8-day old culture eluted from a C18 SPE column
(Waters, Milford, MA) was fractionated on a phenyl-hexyl HPLC column. Analysis of the biofilm-inhibiting fraction by proton NMR
spectrum revealed the presence of three-types of compounds: saturated fatty acids, morpholine, and norspermidine. The active
fraction was further fractionated with a C18 HPLC column; only the sub-fraction that was identified to be norspermidine was active,
whereas the other fractions were inactive. To confirm the results, norspermidine, morpholine, and a few fatty acids (e.g., octanoic
acid and decanoic acid) were purchased from Sigma, and all the commercially purchased compounds were tested for biofilm-inhibiting activity. Only norspermidine showed biofilm inhibition.
Norspermidine Quantification
Because of the absence of UV absorbance of norspermidine at 210 and 280 nm (Merkel et al., 2007) and the three amino groups in the
molecule, it was difficult to develop a simple method for quantifying norspermidine in conditioned medium with conventional UV and
MS methods. Hence, we used Fmoc-Cl (Molnár-Perl, 2003) as a derivatizing agent (Figure S1B).
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. S1
A stock solution of norspermidine (10 mg/ml) was prepared by dissolving it in dry acetonitrile. The norspermidine stock solution
was further diluted with dry acetonitrile to get working solutions ranging from 16 mg to 4800 mg/ml. A 10 mg/ml stock solution of
Fmoc-Cl was prepared in dry acetonitrile. All solutions stored at 4 C and were stable for at least 4 weeks. Into the working solutions
was added DIEA (N,N-diisopropylethylamine). After addition of the stock Fmoc-Cl solution (molar ratio of Fmoc-Cl and norspermidine: 3.5:1), each of the solutions (final concentrations of norspermidine: 0.001–0.4 mM) was mixed 10 min at room temperature
before LC/MS analysis. The derivatives were analyzed by an Agilent LC/MS with an isocratic solvent system of 80% CH3CN
(0.1% formic acid) for 10 min, and then to 100% CH3CN (0.1% formic acid) in 10 min (Flow-rate: 0.5 ml/min; Column: Phenomenex,
Luna, C18, 100 3 4.6 mm, 2.6 m) to obtain a calibration curve by ion count integration at 798 Da, Reaction product of Fmoc-Cl and
norspermidine, [M+H]+, C51H48N3O6, M: NH(Fmoc)-CH2CH2CH2-N(Fmoc)-CH2CH2CH2-NH(Fmoc); Retention time: 10.1 min).
Conditioned medium and pellicles were obtained from 3-day-old and 8-day-old biofilms as well as from norspermidine biosynthetic gene mutants:
(1) Conditioned medium was obtained by subjecting the culture (pellicle and medium) to centrifugation for 5 min at 8,000 rpm. The
resulting supernatant fluid was then filtered through a 0.22 mm filter.
(2) Pellicles were obtained by separating pellicle material mechanically with a spatula from medium. The pellicle material was then
subjected to centrifugation for 5 min at 8,000 rpm, washed in PBS and re-suspended in 10 ml of PBS buffer. The pellets were
then mildly sonicated and the extracellular fraction, which was separated from the cells by centrifugation for 10 min in
8,000 rpm, was analyzed for nospermidine.
For both (1) and (2) above, one milliliter of sample solution was dried, and dissolved in 50 ml dry DMSO (dimethyl sulfoxide). 20 ml of
DIEA (N,N-diisopropylethylamine) and then 3 mg of FMOC-Cl in 30 ml dry acetonitrile were added into the DMSO solution. The derivatized samples were analyzed with an Agilent LC/MS system.
Assays of b-Galactosidase Activity
Cells were cultured in MSgg medium at 37 C in a water bath with shaking. 1 ml of culture was collected at each time point. Cells were
spun down and pellets were suspended in 1 ml Z buffer (40 mM NaH2PO4, 60 mM Na2HPO4, 1 mM MgSO4, 10 mM KCl, and 38 mM
b-mercaptoethanol) supplemented with 200 mg ml-1 of freshly dissolved lysozyme. Suspensions were incubated at 30 C for 15 min.
Reactions were started by adding 200 ml of 4 mg ml-1 ONPG (2-nitrophenyl b-D-galactopyranoside) and stopped by adding 500 ml of
1 M Na2CO3. Samples were briefly spun down. The soluble fractions were transferred to cuvettes (VWR), and OD420 values of the
samples were recorded with a Pharmacia ultraspectrometer 2000. The b-galactosidase specific activity was calculated according
to the equation [OD420/time 3 OD600] 3 dilution factor 3 1000. Assays were conducted at least in triplicates.
Crystal Violet Staining
CV staining was done as described previously except that the cells were grown in 12-well plates (O’Toole and Kolter, 1998). Wells
were stained with 500 ml of 1.0% CV dye, rinsed twice with 2 ml DDW and thoroughly dried. For quantification, 1 ml of 95% ethanol
was added to each well. Plates were incubated for an hour in room temperature with shaking. CV solution was diluted and the OD at
595 nm was measured with Ultraspec 2000 (Pharmacia Biotech).
Dynamic Light Scattering
Exopolysaccharide was diluted to a final concentration of 1, 3, 15, and 30 mg/ml. Light scattering was measured before and after the
addition of norspermidine or spermidine (0.75 mM final concentration). Dynamic light scattering measurements (Berne and Pecora,
1976) were performed by focusing a vertically polarized light (532 nm) onto the sample and collecting the scattered light with
a detector at 90 . Data were collected for 1030 min at 30 s intervals, each with a count rate of (20–150) 3 103 counts/s. The temperature was kept constant at 25 C.
We used the CUMULANT method to analyze the normalized correlation function and extract an average decay rate, Gav. A translational diffusion coefficient Deff was then calculated with the relation Deff = Gav /q2 where q is a wave vector,
4pn
q
sin
q=
l
2
n is the refractive index of the medium, l is the laser wavelength and q is the scattering angle.
Finally, we used the Stokes-Einstein relation to calculate the average hydrodynamic radius, Rav.
Rav = KBT/6p hDeff where KB is the Boltzmann constant, T is the absolute temperature, h is the viscosity of the medium at a given
temperature, and Deff is the particle effective diffusion constant.
Scanning Electron Microscopy
Exopolysaccharide was dissolved in DDW at a final concentration of 10 mg/ml concentration and mixed with norspermidine or spermidine (0.75 mM final concentration). A 5–10 ml drop was then placed on poly-L-lysine-coated 1 3 1 cm2 Si surfaces (wafers
purchased from University Wafers) and kept in a humid environment for 30 min. The Si surfaces were then rinsed with DDW. To
S2 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
capture the native state of exopolysaccharide we kept the surfaces wet at all times and critical-point dried the samples after
exchanging the water with ethanol. The latter step was accomplished by immersing the surfaces in ethanol solutions of increasing
concentrations (from 30%–100% absolute ethanol). Images were obtained with a Zeiss Supra55 Field Emission scanning electron
microscope.
SUPPLEMENTAL REFERENCES
Berne, B.J., and Pecora, R. (1976). Dynamic Light Scattering (New York: Wiley).
Branda, S.S., González-Pastor, J.E., Ben-Yehuda, S., Losick, R., and Kolter, R. (2001). Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci. USA 98,
11621–11626.
Emerson, W.S., and Longley, R.I., Jr. (1963). Diethyl g-Oxopimelate. Organic Syntheses Coll. 4, pp. 302. Annual Vol. 33, pp. 25 (1953).
Gryczan, T.J., Contente, S., and Dubnau, D. (1978). Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis.
J. Bacteriol. 134, 318–329.
Kolodkin-Gal, I., Romero, D., Cao, S., Clardy, J., Kolter, R., and Losick, R. (2010). D-amino acids trigger biofilm disassembly. Science 328, 627–629.
López, D., and Kolter, R. (2010). Functional microdomains in bacterial membranes. Genes Dev. 24, 1893–1902.
Merkel, P., Beck, A., Muhammad, K., Ali, S.A., Schönfeld, C., Voelter, W., and Duszenko, M. (2007). Spermine isolated and identified as the major trypanocidal
compound from the snake venom of Eristocophis macmahoni causes autophagy in Trypanosoma brucei. Toxicon 50, 457–469.
Molnár-Perl, I. (2003). Quantitation of amino acids and amines in the same matrix by high-performance liquid chromatography, either simultaneously or
separately. J. Chromatogr. A 987, 291–309.
O’Toole, G.A., and Kolter, R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30,
295–304.
Sambrook, J., and Russell, D.W. (2001). Molecular Cloning. A Laboratory Manual (Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press).
Wach, A. (1996). PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12, 259–265.
Yasbin, R.E., and Young, F.E. (1974). Transduction in Bacillus subtilis by bacteriophage SPP1. J. Virol. 14, 1343–1348.
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. S3
Figure S1. Detecting Norspermidine and Measuring Its Minimum Inhibitory Concentration, Related to Figure 1
(A) Minimal biofilm-inhibiting concentration of norspermidine, related to Figure 1.
Pellicle formation of strain NCBI 3610 was tested in the presence of various concentrations of norspermidine as indicated.
(B) Detection of norspermidine.1. Norspermidine purchased from Sigma Aldrich was used as a standard for the detection of norspermidine in the biofilm.
Norspermidine was derivatized with Fmoc-Cl and the resulting Fmoc-norspermidine (RT: 10.1 min) was quantified with an Agilent LC/MS system. 2. The UV
spectrum of the reaction product of Fmoc-Cl and norspermidine at 10 min. 3. Positive MS (798 Da) of the reaction product of Fmoc-Cl and norspermidine at
10.1 min. 4. Derivatization reaction of norspermidine with Fmoc-Cl.
S4 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
Figure S2. Effect of a Mutant Blocked in Norspermidine Production and Similarity of the Effect of Norspermidine to the Absence of Exopolysaccharide, Related to Figures 2A and 3
(A) Cells mutant for a homolog of the norspermidine decarboxylase gene yaaO are delayed in pellicle disassembly, Related to Figure 2A. NCBI 3610 (WT), a mutant
for yaaO (IKG624), a mutant doubly deleted for ylmE and racX (IKG55) or a triple mutant for yaaO, ylmE and racX (IKG626) for were grown in 12-well plates and
incubated for 7 days.
(B) Norspermidine treatment phenocopies an exopolysaccharide mutant, related to Figure 3. Shown are 3-day-old cultures of the wild-type (NCBI 3610), an
exopolysaccharide mutant (DepsH; DS76), a TasA mutant (DtasA; FC55), and, as indicated, wild-type and mutant strains grown in the presence of 25 mM
norspermidine.
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. S5
Figure S3. Specificity of Concanavalin A-Texas Red Staining and Lack of Effect of Norspermidine on TasA, Related to Figure 3
(A) Concanavalin A-Texas red stain is largely specific to exopolysaccharide. Related to Figure 3. Fluorescence microscopy was carried out with 3-day-old
standing cultures. Cells of wild-type strain (NCBI 3610) and an eps mutant (DS76) were collected and stained for one hour as in Experimental Procedures. Cells
were imaged with the indicated exposure times. Little or no staining was observed for the mutant except when image brightness was enhanced as shown in the
enlargement or when the cells were stained for 150 min (data not shown). Images were collected with the automated software program SimplePCI.
(B) TasA-mCherry is not released from norspermidine-treated cells. B. subtilis 3610 containing the tasA-mCherry fusion (DR30) was grown without shaking in
a biofilm medium (upper row) or in the same medium applied with norspermidine (50 mM, lower row). Cells were washed in PBS, and visualized by fluorescence
microscopy.
S6 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
Figure S4. Lack of Inhibition of Growth and Gene Expression by Norspermidine, Related to Figure 4
(A) Norspermidine does not inhibit growth of B. subtilis at concentrations that block biofilm formation. Figure related to Figure 4.NCBI 3610 was grown in MSgg
medium applied with norspermidine (100 mM) with shaking or in untreated medium served as a control (NT).
(B) Norspermidine does not inhibit expression of PepsA-lacZ at concentrations that block biofilm formation. Figure related to Figure 4. Strain FC5 (carrying PepsAlacZ) was grown in MSgg medium containing norspermidine (100 mM) with shaking or in untreated medium as control (NT).
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. S7
Figure S5. Structure Activity Relationship Study of Polyamines, Related to Figure 5
The effect of the numbered compounds on pellicle formation by B. subtilis (NCBI 3610).
S8 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.
Figure S6. Structure Activity Relationships for S. aureus and E. coli, Related to Figures 6 and 7
(A) Structure activity relationship study of polyamines in S. aureus. Figure related to Figure 6.The effect of the numbered compounds on the formation of
submerged biofilms by S. aureus strain SCO1. Biofilm formation was visualized by CV staining of submerged biofilms.
(B) Structure activity relationship study of polyamines in E. coli. Figure related to Figure 7. The effect of the numbered compounds on the formation of submerged
biofilms by E. coli strain MC4100. Biofilm formation was visualized by CV staining of submerged biofilms.
Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc. S9
Figure S7. Modeling of Norspermidine Binding to a Neutral Exopolysaccharide, Related to Figure 6
Two PGA stands can be aligned with norspermidine by alternating hydrogen bonds to the acetyl groups of PGA.
S10 Cell 149, 684–692, April 27, 2012 ª2012 Elsevier Inc.