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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. 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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.