Abstract Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

Author Summary Bacteria colonize most environments, including the host by forming biofilms, which are extremely (adaptively) resistant to conventional antibiotics. Biofilms cause at least 65% of all human infections, being particularly prevalent in device-related infections, infections on body surfaces and in chronic infections. Currently there is a severe problem with antibiotic-resistant organisms, given the explosion of antibiotic resistance whereby our entire arsenal of antibiotics is gradually losing effectiveness, combined with the paucity of truly novel compounds under development or entering the clinic. Thus the even greater resistance of biofilms adds to the major concerns being expressed by physicians and medical authorities. Consequently, there is an urgent need for new strategies to treat biofilm infections and we demonstrate in the present study an approach, based on the inhibition of (p)ppGpp by a small peptide, that eradicates biofilms formed by four of the so-called ESKAPE pathogens, identified by the Infectious Diseases Society of America as the most recalcitrant and resistant organisms in our society. The strategy presented here represents a significant advance in the search for new agents that specifically target bacterial biofilms.

Citation: de la Fuente-Núñez C, Reffuveille F, Haney EF, Straus SK, Hancock REW (2014) Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response. PLoS Pathog 10(5): e1004152. https://doi.org/10.1371/journal.ppat.1004152 Editor: Matthew R. Parsek, University of Washington, United States of America Received: February 19, 2014; Accepted: April 14, 2014; Published: May 22, 2014 Copyright: © 2014 de la Fuente-Núñez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R21AI098701 and by a grant from the Canadian Institutes for Health Research MOP-74493. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. REWH holds a Canada Research Chair in Health and Genomics. EFH is supported by a postdoctoral fellowship from the Canadian Institutes of Health Research. SKS would like to acknowledge funding from the Natural Sciences and Engineering Research Council of Canada, the Michael Smith Foundation for Health Research, and the Canada Foundation for Innovation, for funding the NMR spectrometer used in this study. CDLFN received a scholarship from the Fundación “la Caixa” and Fundación Canadá (Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: We have filed a provisional patent application on the use of cationic anti-biofilm peptides (U.S. Patent Application No. 61/870,655) of which CDLFN, EFH and REWH are inventors. This does not alter our adherence to all PLOS policies on sharing data and materials.

Introduction Biofilms are structured multicellular communities of microorganisms associated with surfaces. They have been widely studied, in part because they cause at least 65% of all human infections, being particularly prevalent in device-related infections, on body surfaces (skin and soft tissue, lung, bladder, endocarditis, etc.) and in chronic infections [1], [2]. They represent a major health problem worldwide due to their resistance to host defence mechanisms and to conventional antimicrobials, which generally target free-swimming (planktonic) bacteria [1], [2]. Hence, there is an urgent need to identify compounds that effectively clear biofilm-related infections. Bacteria are known to respond to stressful environmental conditions (such as starvation) by activating the stringent response (SR) [3]. As a consequence, the cell synthesizes two small signaling nucleotides, guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and guanosine 5′-triphosphate 3′-diphosphate (pppGpp), collectively denoted (p)ppGpp [3]. These serve as a second messenger response that is induced by a variety of stress conditions, is highly conserved in both Gram-negative and Gram-positive species [3], [4], regulates the expression of a plethora of genes [3], and is known to play a role in biofilm formation in certain species [5]–[11], although some variability has been observed [6], [7], [9], [12]. Synthetic cationic peptides, derived from natural peptides such as the human cathelicidin LL-37 and the bovine peptide indolicidin [13], have been recently identified as biofilm inhibitory compounds [14]. Anti-biofilm peptides are similar to cationic antimicrobial peptides (which are active against planktonic bacteria), comprising both cationic and hydrophobic amino acids [14], but have substantially different structure-activity relationships. Thus, we previously identified peptides with good anti-biofilm but virtually no activity vs. planktonic bacteria (i.e., very high MIC values) [14], and vice versa. Moreover, certain anti-biofilm peptides are active against biofilms formed by Burkholderia cenocepacia [14], a pathogen that is completely resistant to all antimicrobial peptides in the planktonic state. The broad-spectrum activity of anti-biofilm peptides [14] suggests that they target a biofilm-specific process common amongst bacteria. Given the above, we hypothesized that anti-biofilm peptides exerted their activity by blocking a widespread stress response that contributes to biofilm development, and that this was indeed the stringent response mediated through (p)ppGpp. Here, we have identified a peptide that has very broad spectrum activity against many of the most antibiotic-resistant species of concern in human medicine and provide evidence it acts to promote (p)ppGpp degradation.

Discussion The results described here indicate that an anti-biofilm peptide can exhibit potent broad-spectrum activity due to its ability to depress (p)ppGpp levels in live bacterial cells. Previous studies have suggested that the stringent response might be involved in biofilm formation [5]–[11], although some controversy exists as to whether this may be due to the particular experimental conditions used, particularly flow rate and age of the biofilms examined. The stringent response is induced in reaction to bacterial stresses such as amino-acid starvation, fatty acid, iron or nutritional limitation, heat shock, and other stress conditions. It is signaled by the alarmone (p)ppGpp, and modulates transcription of up to one third of all genes in the cell. Conceptually it is designed to divert resources away from growth and division and toward metabolism in order to promote survival. Its specific role in biofilm formation is not known but it may be involved in initiating and/or perpetuating biofilm development. In addition, the results presented here indicate that it may actually suppress the tendency of biofilms to disperse (Fig. 5), and even promote viability in adhered cells (Fig. S1B). Consistent with its effects on biofilms, in vivo studies have shown that (p)ppGpp-deficient mutants are easily cleared by the host, unable to establish chronic infections, incapable of long-term survival and overall more susceptible to exogenous stresses than their parent strains [18], [19]. Interestingly, various classes of antibiotics are known to induce (p)ppGpp synthesis [20-23; Fig. S3], which in turn leads to antibiotic adaptive resistance [12], [24]. Importantly, recent efforts have identified molecules that target the stringent response [25]. Here we have demonstrated that peptide 1018, which triggers the degradation of the (p)ppGpp signal within the cell, acts as a broad-spectrum biofilm inhibitor. Given the importance of biofilms in human medicine, constituting at least 65% of all infections, and increasing antibiotic resistance which in biofilms is adaptive and broad spectrum, such a peptide offers considerable potential in the fight against the burgeoning resistance to antibiotics. Peptides with biofilm inhibitory activities have been previously identified [13], [14], [26]. However, the mechanism of action by which these peptides selectively target and kill biofilm cells of both Gram-negative and Gram-positive bacteria was previously postulated to involve changes in motility, adherence and quorum sensing [13], [14], which are all species-specific and thus do not satisfactorily explain the action of peptide 1018 against a broad range of pathogens. Here we provide evidence that an anti-biofilm peptide, 1018, potently inhibited biofilm formation and eradicated existing mature biofilms in a broad-spectrum manner, through a direct interaction with ppGpp, which led to its degradation in live bacterial cells. The mechanism of action involves direct contact between peptide 1018 and (p)ppGpp so the peptide must be able to cross the cell membranes to reach the cytoplasm. Previous studies have demonstrated that amphipathic cationic peptides, like 1018, have the characteristics of so-called cell penetrating peptides that are able to freely translocate across membranes [27]. The peptide had at least three effects on biofilms, which might reflect the role of (p)ppGpp in cells. First when added prior to initiation of biofilms it prevented biofilm formation, second it specifically led to cell death in biofilms at concentrations that were not lethal for planktonic (free-swimming) cells (Table 1; Figs. 1 and 2), and third it promoted biofilm dispersal even in maturing (2-day old) biofilms (Fig. 2), effects that were in fact reciprocated in mutants unable to accumulate (p)ppGpp (Fig. 5). We suggest that the ability to cause cell death in biofilms might have been due to inhibition of cell wall biosynthesis and triggering of murine hydrolases, a known tendency for antimicrobial peptides [28]. Critically we propose that this is due in part to the impact of the stringent response in bacteria growing in the biofilm state, since stringent response is known to influence susceptibility to cell wall specific antibiotics, presumably through effects on cell wall synthesis [24], while the lack of ppGpp leads to cell death through the Slt soluble lytic transglycosylase [29]. Consistent with this, both peptide-treated biofilms and (p)ppGpp deficient mutants grown in flow cells demonstrated increased bacterial cell filamentation (Fig. S1B) and cell lysis/death (Figs. 1, 2, S1B and Fig. 5). Previous studies have demonstrated that the structure-activity relationships of anti-biofilm peptides vary substantially from antimicrobial peptides [14] despite certain common features (being amphipathic molecules with excess cationic and hydrophobic amino acids). Consistent with this, 1018 was able to potently inhibit Burkholderia cenocepacia biofilms, despite the fact that this species is completely resistant to all antimicrobial peptides. Thus we have an opportunity to now develop more active peptides that have even more potent anti-biofilm activity. Indeed we have recently started to isolate such peptides and obtained preliminary evidence that they also act by inhibiting the stringent response.

Materials and Methods Bacterial strains Strains utilized included wild-type strains of Pseudomonas aeruginosa PAO1, strain H103, and PA14 and clinical isolates E. coli O157, Salmonella enterica serovar Typhimurium (clinical isolate 14028S), Staphylococcus aureus MRSA (clinical isolate #SAP0017), Klebsiella pneumoniae ATTC 13883 (a colistin-heteroresistant reference strain from American Type Culture Collection, Rockville, MD), Acinetobacter baumannii SENTRY C8 (a polymyxin B resistant blood clinical isolate from the U.S.A. obtained through the SENTRY surveillance system) and Burkholderia cenocepacia genomovar IIIa (Vancouver Children's Hospital clinical isolate 4813). P. aeruginosa PAO1 (p)ppGpp mutant ΔrelAspoT [(ΔrelA (Δ181-2019) ΔspoT (Δ200-1948)] and its complemented strain ΔrelAspoT+SR were a kind gift from D. Nguyen [12]. S. aureus parent strain HG001, its (p)ppGpp mutant HG001 rsh syn (Δ942–950 nt) and the strain complemented with full length rsh were kindly provided by T. Geiger [30]. S. enterica serovar Typhimurium parent strain SL1344 and its (p)ppGpp mutant SL1344 ΔrelAspoT (ΔrelA71::kan rpsL ΔspoT281::cat) were provided by K. Tedin [31]. Escherichia coli parent strain (MG1655), E. coli ΔrelAspoT [ΔrelA:: kan (Δ209-2302) ΔspoT:: cat (Δ700-2355)] deletion insertion mutant and E. coli relA+ (p)ppGpp positive control ptac::relA (pALS10) were also provided by D. Nguyen and obtained as previously described [32], [33]. For the expression of relA in the P. aeruginosa mutant strain PAO1ΔrelAspoT, the pHERD20T plasmid carrying an arabinose-inducible promoter was used [34]. A 3.2 kb DNA fragment containing the relA gene was amplified with primers relAF (5'-GCTAGGATGCCTGCGTAATC-3') and relAR (5'-GAGATCGCCATCGAGGAATA-3') and cloned into a TOPO Zero-Blunt cloning vector (Invitrogen) and then into the pHERD20T vector. This construct was then electroporated into electrocompetent P. aeruginosa PAO1ΔrelAspoT cells. Positives clones carrying the plasmid were selected on LB plus 500 µg/ml of carbenicillin, and relA overexpression upon induction was confirmed by RT-qPCR. In all experiments 0.01% arabinose was used to induce the promoter. Peptide synthesis Peptide 1018 (VRLIVAVRIWRR-NH 2 ) used in this study was synthesized by CPC Scientific using solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry and purified to a purity of ∼95% using reverse-phase high-performance liquid chromatography (HPLC). Peptide mass was confirmed by mass spectrometry. Growth conditions The medium used was generally BM2 minimal medium (62 mM potassium phosphate buffer, pH 7.0, 7 mM [(NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 10 µM FeSO 4 ] containing 0.4% (wt/vol) glucose as a carbon source, except for Staphylococcus aureus HG001 wild-type for which BM2 glucose+0.5% casamino acids (CAA) was used, and Salmonella enterica SL1344 that was grown in Luria Broth. Escherichia coli MG1655 was grown in BM2+0.1% CAA. Minimal Inhibitory Concentration (MIC, MBIC 50 , MBIC 100 ) assays The broth microdilution method with minor modifications for cationic peptides [35] was used for measuring the MIC of peptide 1018. Minimal biofilm inhibitory concentrations leading to 50% decrease in biofilm growth (MBIC 50 ) were obtained using 96-well plate assays and crystal violet staining of adherent biofilms as previously described [14]. The minimal peptide concentrations that completely inhibited biofilm formation (MBIC 100 ) were obtained using flow cells at different input concentrations of peptide. Biofilm cultivation in flow cell chambers and microscopy Biofilms were grown in flow chambers with channel dimensions of 1×4×40 mm, as previously described for 72 h at 37°C [14] in the absence or presence of the desired concentration of peptide 1018. Flow cell chambers were inoculated by injecting 400 µl of an overnight culture diluted to an OD 600 of 0.05. After inoculation, chambers were left without flow for 2 h to enable initial adherence, after which the medium (with or without sub-inhibitory concentrations of 1018) was pumped through the system at a constant rate of 2.4 ml/h. In all cases, after 3 days of growth the flow rate (90 rpm) was increased so as to limit the amount of planktonic and loosely-attached cells within the flow cell chamber. All media used (see above) in flow cell assays supported the planktonic growth of the bacterial species tested, as determined by growth curves (e.g. Fig. S1A). Except where otherwise specified, the concentrations of peptide 1018 used were 10 µg/ml for Pseudomonas aeruginosa, Escherichia coli 0157, Acinetobacter baumannii SENTRY C8 and Burkholderia cenocepacia genomovar IIIa 4813, 20 µg/ml for Salmonella enterica sv. Typhimurium 14028S experiments, 2 µg/ml for Klebsiella pneumoniae ATTC13883, and 2.5 µg/ml for methicillin resistant Staphylococcus aureus MRSA #SAP0017. The different concentrations used correspond to the MBIC 100 of the peptide against the different bacterial species as shown in Table 1. For inhibition studies, peptide was added to the flow-through medium immediately after the initial adherence phase, and maintained for 3 days. For treatment of existing biofilms, bacteria were allowed to develop into structured 2-day old biofilms prior to peptide treatment by addition into the flow cell flow-through medium for the following 24 h. Biofilm cells were stained using the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes, Eugene, OR) or Syto-9 alone prior to microscopy experiments. A ratio of Syto-9 (green fluorescence, live cells) to propidium iodide (PI) (red fluorescence, dead cells) of 1∶5 was used. Microscopy was done using a confocal laser scanning microscope (Olympus, Fluoview FV1000) and three-dimensional reconstructions were generated using the Imaris software package (Bitplane AG). Quantification of the overall biofilm biovolume (µm3) and the percentage of live and dead cell volume were performed using Imaris software as previously described [7], [36]. Experiments were performed at least in triplicate. In experiments looking at the effect of enhanced (p)ppGpp production of biofilm susceptibility to peptides, E. coli strain MG1655 expressing relA from a lac promoter on plasmid pALSl0 [32] was grown in flow cells for 3 days in BM2+0.1% CAA containing 20 µg/ml of 1018 and in the presence or absence of 100 µM isopropyl β-D-1-thiogalactopyranoside (IPTG). P. aeruginosa PAO1 and S. aureus HG001 strains were grown as described above but in the presence or absence of serine hydroxamate (SHX). Dispersal biofilm cell assay Cell counts of live dispersed bacteria from flow cell biofilms were performed using strain P. aeruginosa PA14 grown in BM2 minimal glucose medium. P. aeruginosa PA14 biofilms were grown in the flow cell system for 2 days as described above and treated with 0.8 µg/ml or 10 µg/ml of peptide 1018. To count the dispersed viable cells, 1.5 ml of the output flow was collected at the designated times (time 0 and after 3 and 23 h) and serially diluted 10-fold. One hundred-µl portions from these serial dilutions were then plated onto LB agar plates. Plates were incubated at 37°C overnight, and colony counts were performed to obtain total CFU/ml at each time point. (p)ppGpp measurement by thin layer chromatography Bacteria were grown overnight in modified MOPS minimal medium containing 0.4% glucose, 2 mM phosphate (KH 2 PO 4 ), and 0.2% CAA. The cells were then diluted 1∶20 in the same MOPS minimal medium except containing 0.4 mM phosphate (KH 2 PO 4 ) and 500 µM SHX to induce (p)ppGpp synthesis, in the presence or absence of peptide 1018, colistin, tobramycin or polymyxin B and cells were labelled with 10 µCi/ml 32P for 3 h. For experiments evaluating the ability of the peptide (or different antibiotics) to directly lead to degradation of (p)ppGpp, the cells were induced with SHX and allowed to synthesize (p)ppGpp (for 3 h) prior to peptide treatment. Samples were then extracted with frozen 13 M formic acid by three cycles of freeze-thaw. Aliquots (7.5 µl) of the supernatants were applied to 20×20 cm PEI cellulose TLC plates, resolved with 1.5 M KH 2 PO 4 (pH 3.4) for 4 h. After chromatography, nucleotides were visualized by autoradiography and quantified with a MolecularImager FX PhosphorImager and Quantity One software (Bio-Rad). Unlabeled GTP was spotted on the plates as markers and visualized after chromatography by UV light-induced fluorescence. Nucleotide co-precipitation The ability of 1018 to co-precipitate with nucleotides with varying phosphate content was examined as described previously by Hilpert et al. [37]. Peptide 1018 was mixed separately with ppGpp, GTP, GDP, ATP, ADP or NaH 2 PO 4 in 50 mM Tris buffer at pH 7.4. ppGpp was purchased from TriLink BioTechnologies and all other nucleotides were purchased from Sigma. Nucleotide concentrations ranging from 0.25 to 0.008 mM were mixed in a microtiter plate with 0.25 nmoles of 1018 in a final volume of 100 µl per well. Samples were also prepared containing only 1018 or the nucleotide of interest at each concentration. The co-precipitation of 1018 with a nucleotide resulted in an increase in turbidity, which was quantified by measuring the A 620 using a Powerwave X 340 microplate reader (BioTek Instruments Inc., Winooski, VT, USA). 31P-NMR spectroscopy 31P-NMR spectroscopy was used to evaluate the binding of 1018 to ppGpp and GTP. Samples containing 0.5 mM GTP or ppGpp were prepared in buffer (10 mM Tris, 50 mM NaCl, pH 7.4) and 1 mM phosphate was added as an internal chemical shift reference as well as for quantification. Separate samples containing 0.5 mM 1018 mixed with each nucleotide were prepared in the same way. Additional samples of 0.5 mM ppGpp, GTP and ATP with 1 mM phosphate were also prepared in 6.5 M formic acid for comparison to the samples prepared from the P. aeruginosa PAO1 extracts (see below). All samples contained 10% D 2 O. Each NMR sample was briefly centrifuged on a benchtop centrifuge (∼30 s) to pellet any precipitate that formed and the supernatant liquid was used as the NMR sample. 31P spectra were acquired at 25 °C on a Bruker Avance 500 MHz spectrometer, operating at a 31P frequency of 202.272 MHz. A single pulse experiment, with a 90 degree pulse of 20 µs was used, on a BB 500 probe. 4096 scans were acquired for the pure nucleotide samples while 12288 scans were accumulated for samples that contained peptide. Spectra were processed with an exponential window and line broadening of 50 Hz. To evaluate the differential binding of 1018 to ppGpp or GTP, samples containing an equimolar mixture of ppGpp and GTP (both at 0.5 mM) were prepared in Tris buffer. Peptide 1018 was added to separate nucleotide mixtures to achieve final peptide concentrations of 0.25, 0.5, 0.75 and 1 mM. The samples were again centrifuged to pellet the precipitate and the resulting supernatant was used in the NMR experiments. The experiments were performed as above, but the spectra were processed with a shifted sine bell window only. The phosphorous peak signal intensity resulting from unique chemical shift peaks from either ppGpp or GTP was determined at every concentration of 1018 tested. To examine the effect of 1018 on ppGpp levels in vivo, 3×20 ml cultures of PAO1 (in BM2 media with 0.5% casamino acids) were grown overnight at 37°C in the presence of SHX (500 µM) to induce the production of ppGpp. Following overnight incubation, 1018 was added to a final concentration of 20 µg/ml and the sample was grown for an additional hour at 37°C. For comparison, a separate culture was prepared with no 1018 added. The PAO1 cells were harvested by centrifugation for 20 min at ∼2000×g in a Beckman Coulter Allegra 6 centrifuge. All three bacterial pellets were resuspended in a total of 400 µl H 2 O. To prepare the NMR sample, 400 µl of the bacteria suspension was added to 500 µl of 13 M formic acid and 100 µl of D 2 O. The sample was subjected to three rounds of freezing and thawing using liquid nitrogen and a room temperature water bath. The sample was centrifuged at 4°C and 14000 rpm in a microcentrifuge and 500 µl of the resulting supernatant was used as the NMR sample. Spectra were acquired as described for the pure nucleotide samples but with an accumulation of 24576 scans.

Supporting Information Figure S1. (A) (p)ppGpp mutants exhibited reduced ability to form biofilms in flow cells. Biofilms were grown in flow cells and subsequently imaged and analyzed as outlined in the Methods section. Briefly, bacteria were stained with the all-bacteria Syto-9 stain and analyzed using confocal microscopy. Three-dimensional biofilm reconstructions were generated using Imaris software. (p)ppGpp mutants of the different bacterial species showed decreased biofilm formation in flow cells compared to their parent strains (left panel) using media that supported planktonic growth of both parent and mutant strains in each case (right panel). For assessing planktonic growth, cells were grown in 96-well microtiter plates and growth assessed at 37°C under shaking conditions using a TECAN Spectrofluor Plus. The medium used was BM2 minimal medium glucose for P. aeruginosa ΔrelAspoT and its parent strain. BM2+0.1% CAA was used in the case of Escherichia coli MG1655 and its mutant. LB medium was used for Salmonella enterica SL1344 and its mutant. BM2 glucose+0.5% casamino acids was used to grow Staphylococcus aureus HG001 wild-type and its rsh mutant. (B) Mutations in both genes responsible for (p)ppGpp synthesis as well as treatment with modest amounts (0.8 µg/ml) of peptide 1018 caused filamentation and cell death (as revealed by the uptake of propidium iodide that stains bacteria red) of bacteria grown under biofilm conditions in flow cells. (C) Effect of increasing SHX levels on PAO1 planktonic growth. P. aeruginosa PAO1 was grown in BM2 minimal medium and exposed to increasing concentrations of SHX. The growth of these cultures at 37°C under shaking conditions was monitored with a TECAN Spectrofluor Plus by determining the absorbance at 620 nm for 24 h. https://doi.org/10.1371/journal.ppat.1004152.s001 (TIF) Figure S2. Arabinose did not affect biofilm formation or viable cell dispersal from flow cell biofilms. (A) Addition of 0.01% arabinose to flow-through medium for 3 days or for only the first 2 days of the experiment (conditions identical to those of Fig. 5) did not alter biofilm formation in P. aeruginosa PAO1. Bacteria were stained with the all-bacteria Syto-9 stain and analyzed using confocal microscopy. Three-dimensional biofilm reconstructions were generated using Imaris software. Four independent experiments were performed. (B) Exogenous addition of 0.01% arabinose for 3 days or 2 out of 3 days (as in Fig. 5) did not increase cell dispersal from biofilms. Dispersed cells from 2-day old biofilms were collected and viable cell counts performed 3 and 6 h after addition of arabinose was either discontinued for the last 24 h of the experiment or not. Four independent experiments were performed. Student's t test was used (ns, P>0.05). https://doi.org/10.1371/journal.ppat.1004152.s002 (TIF) Figure S3. Conventional antibiotics did not prevent (p)ppGpp accumulation and did not degrade (p)ppGpp. To evaluate whether antibiotics inhibited (p)ppGpp formation they were added at the same time as SHX and (p)ppGpp pools were observed after 3 h using TLC (left panel). All antibiotics were unable to prevent ppGpp accumulation in fact both tobramycin and polymyxin B increased ppGpp levels (left panel). For degradation experiments, (p)ppGpp levels were allowed to increase by growth of cells for 3 h in the presence of SHX as described in Methods and cells were then treated with the different antibiotics. After 5 and 30 min, levels of (p)ppGpp were determined using TLC. Treatment with colistin and polymyxin B for 30 minutes did not lead to direct degradation of (p)ppGpp (right panel). Accumulation of ppGpp upon polymyxin B treatment was more substantial in the inhibition experiments (left panel) than in the degradation experiments (right panel). https://doi.org/10.1371/journal.ppat.1004152.s003 (TIF) Figure S4. Addition of serine hydroxamate stimulated (p)ppGpp accumulation, which was blocked by treatment with peptide 1018. (A) SHX addition led to (p)ppGpp accumulation in planktonic cells. P. aeruginosa PAO1 was grown overnight in MOPS minimal medium containing 0.4% glucose, 2 mM phosphate (KH 2 PO 4 ), and 0.2% CAA. The cells were then diluted 1∶20 in the same MOPS minimal medium except containing 0.4 mM phosphate (KH 2 PO 4 ). Cells were labelled with 10 µCi/ml 32P for 3 h. (p)ppGpp synthesis was induced with 500 µM serine hydroxamate (SHX). (B) Peptide 1018 blocked (p)ppGpp accumulation in Acinetobacter baumannii and Klebsiella pneumoniae. Anti-biofilm peptide 1018 at 5 µg/ml directly prevented (p)ppGpp accumulation in strains A. baumannii SENTRY C8 and K. pneumoniae ATTC13883 as revealed by thin layer chromatography separation of guanine nucleotides extracted from intact cells. Results shown were obtained using thin layer chromatography and correspond to samples untreated and treated with peptide 1018. https://doi.org/10.1371/journal.ppat.1004152.s004 (TIF) Figure S5. 31P-NMR studies showed that peptide 1018 preferentially bound to ppGpp compared to GTP. (A) Binding of ppGpp and GTP by the anti-biofilm peptide 1018 monitored with 31P-NMR. 31P-NMR spectra were acquired for 0.5 mM samples of GTP or ppGpp in 10 mM Tris pH 7.4 or in 6.5 M Formic Acid (Top panel). Separate samples were prepared containing 0.5 mM nucleotide and 0.5 mM 1018 (Bottom panel). The samples were centrifuged and the supernatant was collected and used as the NMR sample. For the samples prepared in Tris buffer, 1018 precipitated GTP and ppGpp from solution resulting in a significant decrease in the amount of free nucleotide remaining in solution and a large reduction in the phosphorous signals in the 31P NMR spectra (bottom panel). In contrast, no precipitate was observed between ppGpp and 1018 under acidic conditions (6.5 M formic acid). The peaks arising from the nucleotide of interest are indicated while the unlabelled peak corresponds to the 1 mM NaH 2 PO 4 added to the sample as an internal standard. (B) Effect of increasing amounts of 1018 on the 31P-NMR signal intensities arising from ppGpp and GTP in NMR samples containing an equimolar mixture of both nucleotides (0.5 mM each). Peak intensities were measured as a relative value compared to the internal reference peak of 1 mM phosphate at ∼4 ppm. The preferential precipitation of ppGpp over GTP by 1018 is evident from the larger decrease in ppGpp phosphorous signals (at −4.2 and −5.5 ppm) compared to the GTP signals (at −5 and −20 ppm). It should be noted that the ppGpp and GTP phosphorous signals at approximately 9 ppm overlapped with one another and could therefore not be examined in this manner. https://doi.org/10.1371/journal.ppat.1004152.s005 (TIF) Figure S6. Determination of 31P chemical shifts for ppGpp, GTP and ATP under nucleotide extraction conditions and monitoring ppGpp levels in vivo from P. aeruginosa PAO1 cell extracts. NMR spectra of 0.5 mM ppGpp, GTP or ATP in 6.5 M formic acid were collected as described in Figure S5. Each sample also contained 1 mM phosphate as an internal chemical shift reference (peak at ∼1.5 ppm). These spectra were used to establish the chemical shifts of these compounds under the acidic conditions employed to extract the nucleotides from PAO1 cultures. The complete 31P-NMR spectrum of nucleotides extracted from PAO1 cultures induced with SHX is shown in the bottom panel. The ppGpp spectrum had a unique peak at ∼0.6 ppm which did not overlap with phosphorous peaks from either GTP or ATP (indicated with an arrow and a vertical dashed line). This peak was used to evaluate the ppGpp levels in the nucleotides extracted from bacterial cultures. https://doi.org/10.1371/journal.ppat.1004152.s006 (TIF)

Acknowledgments We thank D. Nguyen, M. Cashel, C. Wolz and K. Tedin for providing strains. We also gratefully acknowledge George A. Mackie for his expertise and excellent technical advice. Useful discussions with E.M. Lohmeier-Vogel on 31P-NMR of whole cells are also acknowledged.

Author Contributions Conceived and designed the experiments: CdlFN FR REWH. Performed the experiments: CdlFN FR EFH SKS. Analyzed the data: CdlFN FR EFH SKS. Contributed reagents/materials/analysis tools: CdlFN FR EFH SKS REWH. Wrote the paper: CdlFN REWH.