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Rendueles, O.; Ferrieres, L.; Fretaud, M.; Begaud, E.; Hebomel, P.; Levraud, J.P. and J.M. Ghigo. (2012) A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria. PLoS Pathogens. in press

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FIG S1. Protocol and timeline of axenic zebrafish infection and co-infection used in this study. After fertilization, eggs are sterilized and kept in sterile, autoclaved mineral water at 28°C in vented cap cell culture flasks until 6 dpf. Zebrafish larvae are then transferred one-by-one into 24-well microtiter plates containing 2 ml of water per well. Starting at 4 dpf, larvae are fed every 2 days with axenic T. thermophila till day 15. For longer experiments, in addition to Tetrahymena, larvae were also fed axenic A. salina from 10 dpf onwards. Pathogenic bacteria are added to the water at 6 dpf for 6 h and then larvae are transferred to fresh water. To test the protective effect of potentially probiotic strains, larvae were pre-colonized by commensal bacteria diluted in water at 4 dpf, after hatching.

FIG S2. Inflammation marker expression of gnotobiotic zebrafish larvae upon infection by mild pathogens. qRT-PCR was performed using primers specific to il1b (A), tnfa (B), and il22 (C) (inflammation markers) on RNA extracted from pools of 5 larvae at 3 dpi from germ-free larvae or larvae exposed to E. coli MG1655 (control), E. tarda, A. hydrophila sp. hydrophila or A hydrophila sp. dhakensis at 4 dpf. Levels are expressed relative to the germ-free larvae. Error bars represent 95% confidence intervals from three technical replicates; one representative experiment out of two..

FIG S3. Neutrophil localization upon pathogen infection. Neutrophil infection in germ-free mpx::gfp larvae or mpx::gfp larvae infected with E. coli MG1655 (control) or different pathogens. At 4 days post-infection, larvae were fixed and analyzed by whole-mount immunofluorescence. Neutrophils were detected as GFP-expressing leukocytes (green). A quantification of gut-associated neutrophils in infected larvae is indicated on the right for each group of larvae. Note also GFP-expressing enterocytes as indicated by white arrow.

FIG S4. Impact of identified protective strains on E. ictaluri growth and biofilm formation (A) Biofilm assay: E. ictaluri was mixed in a 1:1 ratio with filtered supernatants of probiotic strains and grown in 96-well microtiter plates at 28°C for 48 h. Microtiter plates were then washed 3 times with water and stained with crystal violet. Biofilm formation was quantified by dissolution of crystal violet and measurement at OD 595 nm. Addition of E. ictaluri’s own supernatant was included as a control. (B) E. ictaluri growth in presence of probiotic supernants: E. ictaluri inoculum was mixed in a 1:1 ratio with filtered supernatant (sn.) from E. coli MG1655, E. coli ED1a-sm, and V. parahaemolyticus and allowed to grow at 28°. OD 600 nm measurements were taken every 30 minutes. Growth of E. ictaluri with its own supernatant was included as a control. The assay was performed twice in microtiterplates, and 12 different wells were monitored for each condition. (C) Broth co-cultures of E. ictaluri with the three identified protective strains. 3ml of BHI medium was inoculated with E. ictaluri alone or with probiotic strain and co-cultures were incubated at 30°C with agitation. Serial dilutions of over-night resulting co-cultures were spotted on BHI+catalase plates in order to obtained isolated colonies (E. ictaluri forms patches rather than individualized colonies in absence of catalase). Plates were incubated at 30°C overnight and E. ictaluri and E. coli MG1655,E. coli ED1a-sm, and V. parahaemolyticus cfu were counted. E. ictaluri was distinguished from co-cultivated bacteria based on its characteristic yellowish colony morphotype. Left panel: E. ictaluri cfu in corresponding co-cultures. Right panel: Corresponding protective bacteria cfu in corresponding co-cultures with E. ictaluri. Results are expressed as mean±SD of three co-cultures for each condition.

FIG S5. Neutrophils redistribution in head and gut. Neutrophils redistribution was quantified by calculating the ratio (neutrophils counts in hematopoietic sites/neutrophils counts in the head and gut) for each larvae. Results are presented as mean+SEM. Statistical significance was calculated between the corresponding pretreated larvae infected and non-infected by E. ictaluri using unpaired two-tailed t-test with Welch's correction (*p<0.05, **p< 0.01, ***p<0.001). One larvae infected by E. ictaluri with a high ratio value was considered as an outlier and excluded from this analysis.

FIG S6. Compared life expectancy upon E. ictaluri infection in germ-free and conventional larvae pre-colonized with various E. coli. Mortality of germ-free (black) and conventional (grey) zebrafish larvae pre-colonized at 4 dpf with E. coli MG1655 and E. coli MG1655 F’, and infected at 6 dpf with E. ictaluri. Mean survival is represented by a large hyphen. Standard deviations are also indicated ( ***p<0.001).

Video S1. Edwardsiella ictaluri colonizes both sides of the lower jaw of zebrafish larvae. Larva analyzed 3 days post-infection by whole-mount immunofluorescence, using an antibody staining bacteria; fluorescence image (red) superimposed to transmission images (gray). Larva Z-stack taken with a confocal microscope and 40x objective. Ventral view with some lateral tilt, anterior to bottom. The first image of the movies provides  a visual help on the top left corner (over the eye) roughly indicating the planes of observation throughout the movie, and a coloured scheme of the cartilages visible in the stack. mc: Meckel's cartilage; pq: palatoquadrate; bh: basihyal; ch: ceratohyal (see Kimmel CB; Miller CT and Moens CB. (2001),Specification and morphogenesis of the zebrafish larval head skeleton. Dev Biol. 15;233(2):239-57.) The yellow line depicts the contour of the fish. Note that figure 3D corresponds to a maximal projection of planes 61 to 75 of the whole stack.

Table S1. Bacterial pathogens used to screen for potential lethal effects in axenic zebrafish larvae.

Table S2. Commensal and probiotic bacteria used to screen for potential protective effect in axenic zebrafish larvae.

Table S3. CFU quantification at 9 dpf of germ-free larvae pretreated with selected probiotics at 4 dpf. Means and standard deviations of the number of CFU recovered from larvae are reported (n=4).

Table S4. qPCR quantification of colonization by E. ictaluri in germ-free and probiotic-preteated  larvae . Shown are mean ± SEM from three pools of three larvae each; data have been normalized to one of the samples from high-dose E. ictaluri.

Table S5. Primers used in this study.

 

Chauhan A.; Lebeaux, D.; Decante, B.;  Kriegel, I.; Escande, M.C.; Ghigo J.M. and C. Beloin. (2012) A rat model of long-term indwelling device reveals the importance of immune system in controlling biofilm establishment and related infections. PloS ONE. In press.

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FIG S1. Increase in bacterial count is correlated with increased luminescence and CFU. Bacteria were grown in liquid medium (LB for E. coli and P. aeruginosa and TSB glucose for S. aureus and S. epidermidis) and samples were harvested over time to evaluate bacterial concentration (, CFU/mL) and relative bioluminescence (, ROI, p/S/cm2/sr). (A-C) For E. coli, P. aeruginosa and S. aureus bioluminescence followed growth and remained relatively high. (D) S. epidermidis showed a marked decrease in bioluminescence over time with increasing CFU and displayed lower levels of bioluminescence than the other bacteria. All the values are mean +/- standard deviation of 3 values.

FIG S2. Surgical implantation of TIVAP in rats. TIVAP were surgically implanted in CD/SD (IGS: Crl) rats. (A) Surgery was performed under laminar air flow and aseptic conditions were maintained throughout the surgical procedure. (B) Rats were briefly kept in an isoflurane chamber to calm down and injected intraperitoneally with a ketamine/xylazine/acepromazine mixture to complete sedation and analgesia before starting the procedure. (C) After shaving and skin disinfection, an incision was made at the dorsal midline. (D) The catheter from the pediatric TIVAP was cut at a 10/11 cm length. (E). A subcutaneous pocket was created and the port carefully inserted before being held intact by sutures (F). (G) An incision was made in the neck area on the ventral side and a Huber needle connected to a tuberculin syringe was inserted into the port. (H) The catheter was tunneled under the skin with the help of a tunneling rod provided with the TIVAP kit. (I) The external jugular vein was exposed and two cotton threads inserted beneath the vein on the proximal and distal sides. The catheter was cut at a slant. (J) A small incision was made in the jugular vein and the catheter inserted into the vein and pushed up to the superior vena cava. (K) Blood reflux was checked by pulling the blood carefully and the TIVAP was flushed with 1X PBS. The catheter was held in place by tying the threads. Suturing of both the dorsal and ventral sides closed the wounds (L) and (M). Lidocaine and betadine were applied to the wounds and rats were allowed to recover for 4 days prior to inoculation. Surgical wounds healed within a week (N) and (O).

FIG S3. Long-term biofilms formed in TIVAP implanted in rats. Several rats (n= 3, for each strain) with implanted TIVAP were monitored for persistent infection over a long period of time. Persistent biofilm growth was observed for E. coli up to 60 days (A), for P. aeruginosa up to 65 days (B) and for S. aureus up to a period of 128 days (C). (D) Rats were sacrificed and TIVAP removed to confirm the presence of biofilms by luminescence.

FIG S4. Mimicking of hematogenous colonization. Central venous catheters were colonized by bacteria from other infection routes, including bloodstream infection. (A) 5x108 CFU/500 µL 1X PBS were injected through the tail vein of TIVAP-implanted rats (n= 6). Photon emission due to bacterial colonization on the TIVAP was monitored and appeared to localize at the catheter tip. (B) Localization of bacterial biofilm on the tip of the TIVAP (n= 2/6) was confirmed by removal on day 3 post intravenous injection and imaging of the catheter. Approximately 104 CFU were detected after resuspension of 1 cm of the catheter tip. Representative images are presented.

FIG S5. Immunosuppression led to fatal biofilm borne infection due to bacteremia and severe organ infection. (A) TIVAP was surgically removed and cells harvested from the catheter (Cath) and port separately and plated for CFU/mL. Data are presented as box-and-whisker plots as previously described in Material and Methods. (B) Peripheral blood was withdrawn by puncturing the retro-orbital sinus on the final day of the experiment and plated for CFU/mL. (C) Organs were aseptically removed on the last day of the experiment, homogenized and plated for CFU/mL. All organs were heavily contaminated (all four pathogenic bacteria) with up to 109 CFU/mL. CFU were estimated on LB agar (E.c., E. coli) or P.a., P. aeruginosa) or TSB agar (S.a., S. aureus or S.e., S. epidermidis) plates. Data are presented as box-and-whisker plots as previously described. Number of rats (n) used in the experiment, n = 4 for each strain.

 

.FIG S6. Exacerbation of immune cells in peripheral blood in systemically infected rats. Blood was collected from the rats by retro-orbital puncture as described in material and methods and analysed using ABC vet automated blood analysis machine. (A) Total leukocyte count for control (uninfected rats, n= 6), E. coli (n= 3), P. aeruginosa (healthy, n= 5; sick, n= 4) and S. aureus (healthy, n= 3 and sick n= 3). (B) Granulocyte count in peripheral blood for control (uninfected rats, n= 6), E. coli (n= 3), P. aeruginosa (healthy, n= 5; sick, n= 4) and S. aureus (healthy, n= 3 and sick n= 3). Statistical analysis was done using one-way analysis of variance (ANOVA) using Graphpad Prism version 5.0c. Except when indicated by connectors statistical comparisons were made with control. p value < 0.05 considered significant, *** (p < 0.0001), ** (p < 0.001) and * (p < 0.05).

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FIG S7. ALT treatment of S. aureus biofilms can lead to systemic infection in animals. Treatment of biofilm colonization in TIVAP-implanted rats with ALT led to systemic infection when not combined with systemic treatment. (A) ALT (0 h) was instilled on day 4 post-biofilm colonization in TIVAP. ALT treatment led to flushing of bacteria into the bloodstream for 30% of treated rats and led to organ infection (n= 3/9). (B) Rat showing systemic infection after 168 h of ALT treatment leading to its death. TIVAP was removed and luminescence measured, confirming persistent biofilm colonization. A representative experiment is shown.

Table S1. Microbiological analysis of TIVAP removed from human patients (n = 279) with suspected catheter- related infection.

Table S2. In vivo relation between ROI (p/s/cm2/sr) and CFU/ml in port of implanted TIVAP at 10 dpi.

Allsopp, L.P. ; Beloin, C., Ulett, G.C. ;Valle, J ; Totsika, M. ; Sherlock, O., Ghigo, J.M. and M.A. Schembri. (2011) Molecular Characterisation of UpaB and UpaC - two new Autotransporter Proteins of Uropathogenic Escherichia coli CFT073. Infection and Immunity 80(1):321-32.

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Table S1. Primers used in this study

 

2011cc

Bernier, S. ; Létoffé, S. ; Delepierre, M and Ghigo J.M.. (2011) Biogenic ammonia modifies antibiotic resistance at a distance in physically separated bacteria. Molecular Microbiology 81:705-716.

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FIG S1. Exposure to a non-toxic ammonia-emitting solution (25 mM NH4+) using the 2-Petri-dish assay increases E. coli BL21 MIC towards tetracycline.

FIG S2. Antibiotic resistance modulation triggered by volatile ammonia in Gram-negative and Gram-positive bacteria. Effect of 24 hours exposure in the 2-Petri dish assay using either H20 (Exp ø), ammonia-emitting 10 mM ammonium solution (Exp 10 mM NH4+) or E. coli spent medium (Exp to E. coli s.m.) on increased resistance to tetracycline in E. coli BL21 (A), P. aeruginosa Lm1 (B), S. aureus Xen 36 (C) and B. subtilis (D).

FIG S3. Evolution of the E. coli polyamine content upon exposure to different active or inactive emitting solutions in the 2-Petri-dish assay. (A) Thin layer Chromatography analysis of putrescine and spermidine in various E. coli recipients upon 24-hr-exposure to controls, wild type, aspC spent media, and 10 mM ammonia-emitting ammonium solution (Exp 10 mM NH4+). (B) Putrescine and (C) spermidine intracellular content quantified from TLC analysis of E. coli cells exposed to increasing concentrations of ammonia-emitting ammonium solutions.

FIG S4. Antibiotic resistance modulation triggered by volatile ammonia in Gram-negative and Gram-positive bacteria. Effect of 24 hours exposure in the 2-Petri-dish assay using either H20 (Exp ø), ammonia-emitting 10 mM ammonium solution (Exp 10 mM NH4+) or E. coli spent medium (Exp to E. coli s.m.) on decreased resistance to kanamycin in E. coli BL21 (A), P. aeruginosa Lm1 (B), S. aureus Xen 36 (C) and B. subtilis (D).

FIG S5. Role of known polyamine targets in ammonia-dependent modulation of antibiotic resistance. Luminescence-based biomass quantification of the growth of different mutants upon exposure to E. coli spent medium. A. Compared biomass increase in E. coli wild type and ompR mutant on tetracycline. B. Compared biomass decrease in E. coli wild type and oppA mutant on kanamycin. The data reported represent the mean (n = 3) ± SD. Statistical analysis: P < 0.05 (*), P < 0.01 (**), P < 0.0001 (***) by one-tailed unpaired Student t-test.

Table S1. Ability of different bacteria to induce distant and aerial modulation of antibiotic resistance..

Table S2. Impact of the addition of different carbon sources on volatile-dependent modulation of antibiotic resistance.

Table S3. Complete list of oligonucleotide primers used in this study.

Rendueles, O. ; Travier, L. ; Latour-Lambert, P. ; Fontaine, T. ; Magnus, J. ; Denamur, E.  and Ghigo J.M. (2011) Screening Escherichia coli species biodiversity reveals new biofilm-associated anti-adhesion polysaccharides. mBio 00043-11

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FIG S1. A. Schematic representation of procedure used to produce biofilm extract from biofilms growth under continuous flow; see also (http://www.pasteur.fr /recherche/unites/Ggb/matmet.html). B. Panel of Gram-negative and Gram-positive bacteria used to test activity of biofilm extracts. a) Representative example of biofilm formation in microtiter plate wells in absence of biofilm extract. b) Representative example of broad-range anti-biofilm activity. c) Representative example of narrow range anti-biofilm activity.

FIG S2. Non-biocidal effect of non concentrated Ec300 and Ec111 biofilm extracts. On S.aureus bacterial lawn grown on LB agar plate (A) or in liquid LB medium in microtiter plate (B).

FIG S3. Activity of E. coli Ec300 polysaccharide compared to PNAG-dependent and PNAG-independent biofilm formation. Biofilm inhibition was determined in the presence of E. coli Ec300 polysaccharide added at 50 mg/ml (final concentration in each well). Experiments were performed at least in triplicate; error bars represent standard deviations of means. Statistical t-tests were used to evaluate significance of biofilm inhibition compared to H2O with *** for P < 0.001. PNAG: poly N-acetyl glucosamine.

FIG S4. Biofilm formation of E. coli Ec300 and ∆galF-hisE mutant. A. Image of a typical microfermentor after 72 h at 37°. B. Relative biofilm formation of each strain.

FIG S5. Comparision of ß-galactosidase activity measurements of lacZ transcriptional fusions at different growth stages (exponential, late-stationary phase and biofilm conditions) of indicated genes. Experiments were performed in triplicate; error bars represent standard deviations of means. Statistical t-tests were used to evaluate the significance biofilm inhibition from various extracts compared to addition of M63B1 with * for P < 0.05, *** for P < 0.001.

FIG S6. Determination of surface contact angle of a drop of water in untreated (dH2O), group 2 capsule (G2CPS) and Ec300p-treated microscopy slides.


Contact angle measurements. Microscope glass slides were washed with detergent and extensively rinsed with distilled water. Washed slide were dried under a laminar flow hood and then coated with 300µl of 100 or 500 µg/ml of ethanol-precipitated cell-free extracts for 5 min and rinsed with destilled water. Four μl drop of water were deposited using a DigiDrop machine on the surface of coated and uncoated slides and contact angle was then measured. For each slide, 5 drops were measured and each drop was measured 3 times

Table S1. Natural E. coli isolates used in this study.

Table S2. Description of genomic contigs potentially involved in biosynthesis of  E. coli Ec300 anti-adhesion polysaccharide.

Table S3. Bacterial strains used in this study.

Table S4. Primers used in this study.

Korea, C. G., Badouraly, R., Prevost, M. C., Ghigo, J. M. and Beloin, C. (2010) Escherichia coli K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities  Environ Microbiol.12:1957-77.

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FIG S1.

 Deletion of the studied operons does not alter biofilm formation.

Biofilm assays were realized on PVC and polystyrene microtiter plates at 30 and 37°C. No major differences were observed between single mutants and wild-type strains, with the exception of ∆fim in LB. A. In LB. B. In M63B1Glu. Values were expressed as percentage of the wild type MG1655 level. The data represent an average of four repetitions for each strain. Error bars represent standard deviations.

FIG S2. Controlled constitutive expression of the studied fimbriae operons.The operon transcript levels of M1655∆fim_kmPcL strains were compared by RT-PCR. Transcripts of exponentially growing, planktonic cells in minimal medium at 37°C were reverse transcribed and amplified by RT-PCR. 16S RNA levels were used as internal controls of the quantity of RNA used for RT-PCR. +RT, in the presence (positive control) or -RT, in the absence of reverse transcriptase (negative control). Bands of proportional intensity can be observed for each gene

FIG S3. Microscopic observation of ∆fimPcL strains.TEM images were taken from samples of MG1655∆fim_kmPcL strains grown in minimal medium. a-c: Type 1 fimbriae can be observed for the MG1655 strain and positive control MG1655kmPcLfim (arrows), whereas no fimbriae were observed for negative control MG1655∆fim. d-e: although no filamentous structures were detected on the surface of the MG1655∆fim_kmPcLsfm strain (d), equally separated small size afimbrial protrusions were observed at the cell surface at higher magnification (arrows) (e). f-i: No structures were observed on the surface of strains constitutively expressing ybg, yfc, yra and yeh operons

FIG S4. Identification of the major subunits of ycb, ybg and yeh fimbriae. Putative fimbriae expressed on the surface were extracted either by heat-shock extraction (A) or by mechanical extraction (B, C) of surface and membrane-attached proteins followed by SDS-PAGE analysis. In (B), fimbriae extracts were treated by 8M urea. Profiles were compared with total cell extracts of each strain in order to exclude the possibility of cellular lysis. When compared to wild-type MG1655∆fim extract, specific extracellular proteins were detected in strains constitutively expressing fim (A) but also ycb (A, B, C), yeh (B) and ybg (C). N-terminal microsequencing confirmed that the isolated proteins are, respectively, FimA (18kDa), YcbQ (apparent molecular mass of 18 kDa), YehD (apparent molecular mass of 18 kDa) and YbgD (apparent molecular mass of 24 kDa). In (B), single and double asterisks represent identical proteins present in the three extracts. Because of the urea treatment of the extracts and the presence of high quantiy of YcbQ or YehD, the migration of these proteins is modified in the different samples.

FIG S5. Constitutive expression of ycb interferes with the Ag43-dependent aggregation phenotype. The different strains constitutively expressing each of the 7 fimbriae operons in a ∆oxyR background were grown overnight in LB at 37°C and at 30°C (for ycb and yad) (A). After growth, cells were diluted and adjusted to an OD600 of 2.5 in a 3-mL volume and autoaggregation of each strain over 24 h at room temperature was evaluated by visual estimation. The presence of surface structures on the MG1655kmPcLycboxyR surface inhibited physical Ag43-mediated interactions between bacteria. To a lesser extent, constitutive expression of the yad operon in LB led to an intermediate phenotype, whereas none of the other putative fimbriae altered the Ag43 autoaggregation pattern. Immunodetection of the Ag43 protein by western blot (B) or by immunofluorescence (C-D) in the strains constitutively expressing ycb and yad. (B) MG1655, MG1655∆oxyR, MG1655∆oxyRflu, MG1655kmPcLycboxyR and MG1655kmPcLyadoxyR strains were grown overnight in LB at 37°C. Total protein extracts were prepared and run onto a 10% SDS-PAGE before being transferred to nitrocellulose and revealed with an antiserum recognizing the a-domain of Ag43. MG1655kmPcLycboxyR (C) and MG1655kmPcLyadoxyR (D) strains were grown overnight in LB at 37°C and were fixed without permeabilization on immunofluorescence slides. Bacterial DNA was revealed using DAPI coloration (in blue). The presence of the Ag43 at the cell surface of the bacteria was revealed using an antiserum against the a-domain of Ag43 and a secondary antibody coupled to Alexa 488 (in green). The Ag43 protein is normally exposed to cell-surface upon expression of both ycb or yad operons.

FIG S6. An hns mutant produced multiple fimbrial structures. TEM images were taken from samples of MG1655∆fim (a) and MG1655∆fim_∆hns strains (b-g) grown in minimal medium. a: No fimbriae were observed for negative control MG1655∆fim. b-g: different types of fimbriae are observed when hns is deleted (arrows). Some fimbriae are rather flexible, whereas others seem more rigid (see, for example, the e panel). As observed when the yad operon was constitutively expressed, some flexible fimbriae appeared to join to form bundles of different thicknesses

Table S1. Summary of the characteristics of the identified fimbrial operons.

Table S2. Primers used in this study.

Table S3. List of primers used for semi-quantitative PCR.

Le Quéré, B and J.M. Ghigo (2009). BcsQ is an essential component of the Escherichia coli cellulose biosynthesis apparatus that localizes at the bacterial cell pole.’ Molecular Microbiology 72(3), 724–740

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FIG S1. Inactivation of yhjQ does not lead to a filamentation phenotype in E. coli.

Light microscopy of wild-type E. coli 1094 (a), MG1655 (b) and C600 (c), and of their corresponding ∆yhjQ mutants (a’, b’ and c’), in which a non-polar inactivation of yhjQ is performed by replacing its coding sequence on the 1094 by the kmRExTET cassette with its own ribosome-binding site in front of bcsA (Fig. 2Ad). Strains were cultured with aTc added to ensure the transcription of the downstream genes. No filamentation was observed with of without aTc.

FIG. S2. Impact of the induction of a GFP-YhjQ fusion on s32 activity

A.  Fluorescence at 612 nm of 1094 kmRExTETrbs-gfp-yhjQ with rfp under the control of the ibpAB promoter (pibpA-rfp). Cultures were conducted in LB until OD600 reached ~4 (stationary phase) and dispatched in a 96-wells microtiterplate (7 samples per condition tested). First, the GFP-YhjQ fusion was induced with various doses of aTc. Second, the same strain was transformed with p∆709 (empty plasmid), p1∆SS (coding for a MalE protein without signal sequence) and p31∆SS (coding for the MalE31 variant promoting inclusion bodies formation, without signal sequence). The plasmids were induced with 0.2% maltose. Data were normalized to the appropriate control, aTc 0 for aTc induction, p∆709 for plasmids. Error bars represent standard deviation. The significant differences observed (p-value< 0.05) are reported on the graph (* p-value < 0.05, ** p-value < 0.01, *** p-value <0.005). B. GFP-YhjQ localization induced with 20 ng/ml aTc: strain 1094 kmRExTETrbs-gfp-yhjQ with rfp under the control of the ibpAB promoter was cultured in LB with 20 ng/ml aTc until OD600 reached 1. Phase contrast microscopy: (a); epifluorescence microscopy: GFP (b), DAPI (c), DAPI and GFP (d).

FIG. S3. Correlation between GFP-YhjQ labeled-pole and cellulose production

Epifluorescence microscopy of 1094 amp.gfp-yhjQ, performed as in Fig. 6D. Cellulose is labeled via CBM 29-1-2 and appears in red, GFP fluorescence in green. Twenty different fields with individual cellulose-producing bacteria are presented .

Supplementary video. Tri-dimensional reconstitution of a cellulose-producing bacteria.

Three-dimensional reconstruction from transmission electron microscopy (TEM) of E. coli 1094 wild type with cellulose labeling by CBM 29-1-2 and immunogold. Series of pictures corresponding to slices of the same sample were taken in TEM, aligned using Photoshop CS and processed with the IMOD software. The grey volume represents E. coli 1094 surface. The blue dots account for the gold particles bound to antibodies targeted against cellulose-binding modules.

Table S1. Primers used in this study.

Beloin, C.; Houry, A;, Froment , M. ; Ghigo J.M. and N. Henry. A short time scale colloidal system reveals early bacterial adhesion dynamics and  adhesin-dependent behaviours. PLoS Biology July 2008 | Volume 6 | Issue 7 | e167.

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Figure S1: Surface charge detection by flow cytometry..

Figure S2: GFP+ and white E. coli mixture in FCM analysis..

Figure S3: Fluorescence signal calibration..

Figure S4: MG1655gfpompR234 and MG1655gfpcsgA surface colonization.

Figure S5: Surface charge inversion effects..

Table S1. Primers used in this study.

These supporting information are also available on the PLoS Biology web site at

http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0060167#toclink6

Valle, J. ; Mabett, A.N.; Ulett, G.C.; Toledo-Arana, A.; Wecker, K.; Totsika, M.; Schembri, M.A.; J.M. Ghigo. And C. Beloin, C. UpaG, a new trimeric autotransporter adhesin in uropathogenic Escherichia coli. (2008) J Bacteriol 190(12):4147-61

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Figure S1: upaG is poorly expressed in vitro.

Figure S2: Analysis of fibronectin content is protein lysates of T24 and HeLa cells..

Table S1. Primers used in this study.

Table S2. Natural E. coli isolates used in this study These strains come from collections described in:

Picard, B., J. S. Garcia, S. Gouriou, P. Duriez, N. Brahimi, E. Bingen, J. Elion, and E. Denamur. 1999. The link between phylogeny and virulence in
Escherichia coli extraintestinal infection. Infect. Immun. 67:546–553..

Ochman, H., and R. K. Selander. 1984. Standard reference strains of Escherichia coli from natural populations. J Bacteriol 157:690-3.


This supplementary material is also available on the J. bacteriol. web site at http://jb.asm.org

Valle, J. : Da Re, S., Schmid. S. ; Skurnik, D. ; D’Ari, R. and Ghigo J.M. The amino-acid valine is secreted in continuous-flow bacterial biofilms. J. Bacteriol. 190:264-7

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Figure S1: Inhibitory activity of pure exogenous valine..

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Inhibitory activity of the CFT073 in biofilm supernatant (In-biofilm S) that contains 12 mg/ml of valine and pure exogenous valine (15 mg/ml) on overlayers of MG1655 cells (upper panel) and MG1655DlivH cells (lower panel). The mall halo observed in CFT073 in-biofilm S. on overlayers of DlivH cells is produced by microcine H47
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Figure S2: E. coli strain Ec. 057 is ilvG.

A. Alignment of DNA ilvG sequences of MG1655ilvG+ and E. coli strain Ec. 057. Numbering above indicate base number from the ATG translation start according to ilvG+ sequence. Differences (bases deletion and insertions) are boxed. B. Alignment of the IlvG protein sequence (corresponding to the above DNA sequences) of MG1655ilvG+ and E. coli strain Ec. 057. Stop codon is boxed.

Figure S3: Exogenous valine does not affect biofilm formation of several E. coli strains..

Effect of in biofilm supernatant purified from CFT073DkpsD (S.CFT073DkpsD) and CFT073DkpsDDsspA (S.CFT073DkpsDDsspA)on biofilm formation in micro-fermentors of several E. coli strains.

Table S1. Primers used in this study.

Table S2. Natural E. coli isolates used in this study compiled from:

Escobar-Paramo, P., A. Le Menac'h, T. Le Gall, C. Amorin, S. Gouriou, B. Picard, D. Skurnik, and E. Denamur. 2006. Identification of forces shaping the commensal Escherichia coli genetic structure by comparing animal and human isolates. Environ Microbiol 8:1975-84.

Ochman, H., and R. K. Selander. 1984. Standard reference strains of Escherichia coli from natural populations. J Bacteriol 157:690-3.

Skurnik, D., R. Ruimy, A. Andremont, C. Amorin, P. Rouquet, B. Picard, and E. Denamur. 2006. Effect of human vicinity on antimicrobial resistance and integrons in animal faecal Escherichia coli. J Antimicrob Chemother 57:1215-9.


Some of this supplementary material is also available on the J. bacteriol. web site at http://jb.asm.org

 Da Re, S., Le Quere, Ghigo, J.M. and C. Beloin .(2007). Tight modulation of Escherichia coli bacterial biofilm formation through controlled expression of adhesion factors. Appl. Environl Microbiol. In press.

Figure 1S


Figure 1S. Impact of constitutive expression of flu on the ability of commensal E. coli to compete with enteroaggregtaive E. coli 55989 for biofilm formation.
Strains E coli 55989latt.amp.gfp, an enteroaggregative strain marked with GFP and ampicillin resistance genes inserted at the latt site, E. coli MG1655Dflu, where the flu gene was replaced by a kanamycin resistance gene, and E. coli MG1655kmPcL-flu, where flu expression is triggered by the PcL cassette associated to a kanamycin resitance gene, were grown overnight in M63B1 minimal medium with glucose 0.4% as a carbon source. These cultures were adjusted to OD600 = 1 by dilution and mixed in a 1:9 ratio of 55989 versus MG1655 as follows: E coli 55989latt.amp.gfp + E. coli MG1655Dflu and E coli 55989latt.amp.gfp + E. coli MG1655kmPcL-flu. This ratio based on OD600 was checked by serial dilution and plating of the two starting mixes on LB (total population counts), LB+ampicillin (55989latt.amp.gfp counts) and LB+kanamycin (MG1655 derivatives counts). For each mixed culture, six microfermenters were inoculated by dipping the spatula for 2 minutes in 15 mL. The mixed biofilms were allowed to grow at 37°C for 24h, with a constant flux of M63B1 supplemented with 0.4% glucose. The biofilms grown on the spatula were resuspended in 10mL M63B1, serially diluted and plated on LB, LB+ampicillin and LB+kanamycin. The results are expressed as the ratio of E. coli 55989 CFU relative to E. coli MG1655 CFU in the 24h-old biofilms. Error bars represent the standard deviation of the 55989:MG1655 CFU ratios calculated for each fermenter. The statistical significance of the difference between the ratios 55989 latt.amp.gfp: MG1655 Dflu and 55989 latt.amp.gfp: MG1655 kmPcL-flu was tested using a t-test. The two ratios were found statistically different with a p-value = 6.45.10-5

This supplementary material is also available at http://www.

Valle, J. : Da Re, S., Henry N. ; Fontaine, T.; Balestrino, D.; Latour-Lambert, P.; and Ghigo J.M. (2006) Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide. Proc Natl Acad Sci U S 103:12558-12563.

Figure 1S
Table 1S
Table 2S

Figure S1. Correlation between anti-biofilm activity and group II capsule.
Biofilm formation of E. coli MG1655 F’ and 1091 strains, and of the S. aureus 15981 strain cultured with: (A) supernatants of E. coli exhibiting anti-biofilm activity (see Table 1S) (B) supernatants of CFT073, U-9, U-15 and their respective kpsD mutants. (C) Biofilm formation in microfermentor of UPEC strains CFT073, U-9, U-15 (black) and their respective kpsD mutants (grey) grown in M63B1glu, and kpsD mutants grown in media supplemented with their corresponding wild-type supernatant (white). Biofilms were grown for 36 h at 37°C. Error bars represent standard deviation of the mean.

Table 1S. Strains used in this study

Table 2S. Primers used in this study.

This supplementary material is also available at http://www.PNAS.org

Da Re, S. and Ghigo, J. M. (2006) A CsgD-Independent Pathway for Cellulose Production and Biofilm Formation in Escherichia coli J Bacteriol.188:3073-87.

Figure 1S
Figure 2S
Table 1S
Table 2S

Fig. 1S. Cellulose production is responsible for clumps formation and is the main component of the biofilm matrix of E. coli strain 1094.
A. Standing tubes of o/n cultures of strains 1094 and 1094bcsC grown in minimum medium at 30°C on a rotating wheel.
B. Clump disruption: Same as A with strains 1094, 1094csgD and 1094csgD-pgi grown in the absence or presence of 10 µg ml-1 of purified recombinant CelD endo-b-1,4-glucanase of C. thermocellum. The growth rate of the pgi mutant is severely impaired in minimal media, thus explaining the weak turbidity of the 1094csgD-pgi culture in the presence of cellulase compared to the wild type.
C. Biofilm disruption: A 24 hours old biofilm of strain 1094 and TG1 formed on a pyrex spatula were incubated in a standing tube containing 50 mM sodium citrate buffer pH 7.5 without or with 0.1% of commercial cellulase (Sigma ref: C-9422) for 16 hours at 37°C.

Fig. 2S. Transcription of yedQ and adrA in strain 1094 in different environmental conditions.
A and B. yedQ expression was studied in the wild type E. coli 1094 and the 1094rpoS mutant by measuring the b-galactosidase activity of a yedQ:lacZ fusion carried on the pRSpyedQ::lacZ plasmid. The cloning vector pRS415 was used as a negative control in both strains. The measurements were done at least in triplicate in exponential (Exp) and stationary (Stat) phases, as indicated on the x-axis, at 37°C in LB medium (A) and at 30°C in LB and M63B1 medium supplemented with 0.4% glucose (B). Light grey bars: b-galactosidase activity in 1094; dark grey bars: b-galactosidase activity in 1094rpoS.
C and D. yedQ and adrA expression were compared in strain E. coli 1094 by measuring the b-galactosidase activity of a yedQ:lacZ and a adrA:lacZ fusions carried respectively on the pRSpyedQ::lacZ and pRSpadrA::lacZ plasmids. The cloning vector pRS415 was used as a negative control. Experiments were done at least in triplicate at 37°C in Exp. and Stat. phases, as indicated on the x-axis, at 37°C in LB (C) and at 30°C in LB and M63B1 medium supplemented with 0.4% glucose (D). White bars: pRS415 b-galactosidase activity; light grey bars: pRSpyedQ::lacZ b-galactosidase activity; dark grey bars: pRSpadrA::lacZ b-galactosidase activity.

Table 1S. CF, CR and biofilm phenotypes analysis in different E. coli strains.

Table 2S. Primers used in this study.

This supplementary material is also available at http://jb.asm.org/cgi/content/full/188/8/3073/DC1

C. Beloin, K. Michaelis, K. Lindner, P. Landini, J. Hacker, J. M. Ghigo and U. Dobrindt. (2006) The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli J Bacteriol.188:1316-31.

Please note that Dr. Uli Dobrindt is the corresponding author on this publication
Dr. Ulrich Dobrindt
Institut fuer Molekulare Infektionsbiologie
Universitaet Wuerzburg
Roentgenring 11
97070 Wuerzburg
Germany

Tel.: ++49 (0)931 312155
Fax: ++49 (0)931 312578
E-mail: ulrich.dobrindt@mail.uni-wuerzburg.de
Table 1S
Figure 1S
Figure 2S Figure 3S Figure 4S

Fig. 1S. Autoaggregation phenotype of E. coli K-12 cells containing the rfaH mutation. Cells were grown overnight in M63B1 0.4% glucose before being diluted to the optical density of 3 in 3 ml of medium. A. Optical density of the upper part of the standing tube was measured during the day and after 24 hours. Error bars correspond to averaging of three independent experiments. B. Images of the standing tubes were captured after 24 hours at room temperature. A representative experiment is shown.

Fig. 2S. Detection of agn43 orthologs in the genome of E. coli strain 536 and derivatives by Southern hybridization. Genomic DNA of UPEC strain 536 was digested with EcoRI prior to DNA fragment separation by agarose gel electrophoresis and DNA transfer to a nylon membrane. The blot was then hybridized with a 32P-labelled 590-bp DNA probe obtained by PCR using the primers (KM-Ag43_fwd, KM-Ag43_rev). The resulting PCR product is complementary to a conserved part of  the passenger domain of agn43.

Fig. 3S. Analysis of dam and oxyR transcript levels of uropathogenic E. coli strain 536 and K-12 strain MG1655. The corresponding wild type strains and their rfaH mutants were cultivated under different growth conditions (exponential (log) and stationary (stat) growth phase of planktonic cells as well as from biofilms) and the dam and oxyR transcript levels were compared by RT-PCR. Biofilms were harvested after 24 h (bio24) or 48 h (bio48) of growth.

Fig. 4S. LPS is a major adhesion factor to abiotic surfaces of strain 536. A. Biofilm development of strain 536 and its derivatives was compared in microfermentors after 48 h of growth at 37 °C in M63B1 glucose medium. A representative experiment is shown. B. The average of at least three experiments was plotted in the histogram. Error bars represent standard errors of the mean. The level of biofilm formed by wild type strain 536 was set to 100 %. C. The LPS pattern of the strains was compared.

Roux, A, Beloin, C. and Ghigo, J.M.(2005) A combined inactivation/expression strategy to study gene function in physiological conditions: application to the identification of new adhesins in E. coli. J. Bacteriol.187:1001-1013.

Table 1S

C. Beloin, J. Valle, P. Latour-Lambert, P. Faure, M. Kzreminski, D. Balestrino, J. A. Haagensen, S. Molin, G. Prensier, B. Arbeille and J. M. Ghigo. (2004.) Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression Mol Microbiol.51:659-74.

Figure S1
Figure S2
Figure S3
Table S1
Table S2
Table S3
Table S4
Table S5

Fig. S1. COG functional classes for genes underexpressed in TG1 biofilm versus exponential growth phase.

Fig. S2. Functional profiling of mature E. coli biofilm: biofilm formation in microfermenters.

Fig. S3. Functional profiling of early steps in E. coli biofilmformation.

Table S1. Genes overexpressed in E. coli TG1 biofilm versus exponential growth phase.

Table S2. Genes underexpressed in E. coli TG1 biofilm versus exponential growth phase.

Table S3. Genes overexpressed (≥2) in E. coli TG1 biofilm versus both exponential and stationary growth phase.Table S4. Inactivation of the genes described in this study and TG1gfp strain construction: primers used in the linear DNA, three-step PCR inactivation protocol.

Table S5. Primers used for the Q-RT-PCR experiments.

This supplementary material is also available from http://www.blackwellpublishing.com/products/journals/suppmat/mmi/mmi3865/mmi3865sm.htm

Additional material; Biofilm versus stationary growth phase analysis:

J. Valle, A. Toledo-Arana, C. Berasain, J. M. Ghigo, B. Amorena, J. R. Penades and I. Lasa. (2003.) SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus Mol Microbiol.48:1075-87.

Please note that Dr. Inigo Lasa is the corresponding author on this publication
Inigo Lasa
Associate Professor of Microbiology
Instituto de Agrobiotecnologia y Recursos Naturales
Universidad Publica de Navarra
Pamplona-31006
NAVARRA
SPAIN
Phone 34 948 168007
Fax 34 948 232191
email: ilasa@unavarra.es

Table 1S

Table S1. Sequence analysis of transposon-tagged genes of selected biofilm deficient mutants.

This supplementary material is also available from http://www.blackwellpublishing.com/products/journals/suppmat/mole/mole3493/mmi3493sm.htm

C. Solano, B. Garcia, J. Valle, C. Berasain, J. M. Ghigo, C. Gamazo and I. Lasa. (2002.) Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose Mol Microbiol.43:793-808.

Please note that Dr. Inigo Lasa is the corresponding author on this publication
Inigo Lasa
Associate Professor of Microbiology
Instituto de Agrobiotecnologia y Recursos Naturales
Universidad Publica de Navarra
Pamplona-31006
NAVARRA
SPAIN
Phone 34 948 168007
Fax 34 948 232191
email: ilasa@unavarra.es

Figure S1

Fig. S1. Biofilm formation phenotypes on PVC microtitre plates. A comparison of 48 h biofilms made by S. enteritidis wild type (3934) and representative mutants from group I (pgm–), group II (cytR–) and group III (yhjL–). The biofilm is concentrated at the interface between the air and the liquid medium (indicated by an arrow).

This supplementary material is also available from http://www.blackwell-science.com/products/journals/
suppmat/mole/mole2802/mmi2802sm.htm

J. M. Ghigo. (2001.) Natural conjugative plasmids induce bacterial biofilm development Nature.412:442-5.

Figure 4S
Figure 5S
Figure 6S
Figure 7S

Figure 4S : A Kinetics of biofilm formation of strain TG1 carrying a GFP-expressing plasmid pGFPmn2. (Top): biofilm at different times after inoculation. Size bars: 10 µM (Bottom) Epifluorescence photomicrographs of the corresponding Pyrex slides. B Scanning laser confocal photomicrographs of TG1 (pGFPmn2). (Top) Schematic indicating the observation point. (Bottom) Z-axis.

Figure 5S : Kinetics of surface adhesion in the F- and F+ E. coli strains.Compared cell density of strain MG1655 Ftet and MG1655 after 5; 10 and 24 hours of culture in microfermenters. Transmitted light microscopy of the Pyrex slide stained with crystal violet. Size bars : 10 µm.

Figure 6S : Natural conjugative plasmids promote biofilm formation.Representative examples of the biofilm promoting ability of natural conjugative plasmids.  (Left) Plasmid-free E. coli F- strain BM21. Incompatibility groups of the presented plasmids are indicated.

 Figure 7S: Derepression of conjugation ability of natural conjugative plasmids.Comparison of biofilm formation ability of strain E. coli BM21 carrying natural conjugative plasmids inoculated alone (left) or co-inoculated 24H after the initial inoculation of strain E. coli MG1655. Conjugative plasmids are described in Table 1.

This supplementary material is also available at http://www.nature.com).