Supporting Information

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1 Supporting Information Beauregard et al /pnas Fig. S1. Model depicting the regulatory pathway leading to expression of matrix genes. The master regulator Spo0A can be directly or indirectly phosphorylated by the five histidine kinases, KinA KinE. When the level of phosphorylated Spo0A (Spo0A P) reaches a threshold, sini transcription is activated. SinI acts as an anti-repressor that binds the repressor SinR, allowing the expression of matrix genes. Fig. S2. Only a small subset of B. subtilis cells grown in minimal salts nitrogen glycerol (MSNg) medium express matrix genes. Wild-type (3610) cells harboring P tapa -yfp were inoculated in MSNg medium and imaged after 24 h. Shown are overlays of fluorescence (false-colored green) and transmitted light (gray) images. 1of6

2 Fig. S3. B. subtilis does not colonize hair in minimal salts nitrogen (MSN) medium. Wild-type (3610) cells constitutively expressing yfp were incubated in the presence of sterilized human hair and incubated for 24 h. Overlay of fluorescence (false-colored green) and transmitted light (gray) images is shown. Fig. S4. Arabinogalactan, pectin, and xylan support growth of B. subtilis. Growth curve of cells shaken at 37 C in MSN with the indicated compounds as a sole carbon source. Note that the initial decrease in the xylan curve is due to the fact that xylan displays some absorbance at OD 600 and that, as the bacteria break down the xylan, there is a decrease in OD 600 until growth of bacteria surpasses xylan breakdown. 2of6

3 Fig. S5. Galactose, Arabinose, Fucose, Xylose, and Glucuronate do not induce biofilm formation. (A) Pellicle formation of wild-type cells in MSNc. The indicated mono- or polysaccharides were added at a final concentration of 0.5% at the onset of the assay. AG, Arabinogalactan. Images are top-down view of wells and were taken after 24 h at 30 C. (B) Pellicle weight assay of wild-type cells in MSNc. The indicated mono- or polysaccharides were added at a final concentration of 0.5% at the onset of the assay. Analysis of variance revealed a significant main group effect between the conditions used [F(6, 21), P < 0.001]. Tukey s post hoc test revealed that only galacturonic acid, arabinogalactan, and pectin (marked with asterisks) showed greater pellicle mean mass compared with MSNc alone, and that galacturonic acid was statistically different then pectin. Fig. S6. Plant polysaccharides induce pellicle formation at a concentration of 0.5%. Pellicle weight assay of wild-type cells in MSNc. The indicated mono- or polysaccharides were added at the indicated final concentration of 0.5% at the onset of the assay. Two-way analysis of variance revealed a significant main group effect between the conditions used [F(2, 27), P < 0.001]. Tukey s post hoc test revealed that concentrations of 0.05% and 0.1% of any sugars showed lesser pellicle mean mass compared with concentration of 0.5%. 3of6

4 Fig. S7. The double deletion of KinC and KinD partially affects plant colonization. Mutant strains constitutively expressing YFP were coincubated with 6-d-old seedlings of A. thaliana. Colonization of the root was observed after 24 h. Overlays of fluorescence (false-colored green) and transmitted light (gray) images are shown. Pictures are representative of at least twelve independent roots. (Scale bars: 50 μm.) Fig. S8. Galactose rescues biofilm formation of a gale mutant in minimal salts glutamate glycerol (MSgg) medium. Deletion mutants for gale, galkt, or both were assayed for pellicle formation in the biofilm-inducing medium MSgg with or without the addition of galactose. Top-down view of pellicle formed by the indicated strains after incubation for 24 h at 30 C. 4of6

5 Fig. S9. Xyloglucan but not carboxymethylcellulose induces pellicle formation. Pellicle weight assay of wild-type cells in MSNc. The indicated polysaccharides were added at the indicated final concentration of 0.5% at the onset of the assay. CMC, carboxymethylcellulose. Two-way analysis of variance revealed a significant main group effect between the conditions used [F(3, 28), P < 0.001]. Tukey s post hoc test revealed that xyloglucan showed a greater pellicle mean mass compared with MSNc alone, and that xyloglucan was statistically different from AG. Fig. S10. The anti-sigma factor rsgi and its cognate sigma factor sigi are not involved in responding to plant polysaccharides. Top-down view of pellicle assay in which the indicated mutant cells were incubated for 24 h at 30 C in the presence of arabinogalactan (AG), pectin, or xylan in MSNc medium. 5of6

6 Table S1. Strains used in this study Strain Genotype Reference/source NCIB3610 Wild type. Undomesticated strain Lab stock CA018 amye::p tapa -yfp (spec) (1) PB133 amye::p hyperc1o1 -yfp (spec) Edgardo Sanabria-Valentín HV1142 amye::p spac -cfp (spec) This study PB178 eps::tet tasa::kan amye:: Phyperc1o1 -yfp (spec) This study PB228 eps::tet amye::p hyperc1o1 -yfp (spec) This study PB229 tasa::mls amye::p hyperc1o1 -yfp (spec) This study PB251 tasa::kan amye::p spac -cfp (spec) This study PB240 spo0a::mls amye::p hyperc101 -yfp (spec) This study PB177 sini::kan amye::p yqxm -yfp (spec) This study SSB43 spo0a::mls (2) ZK4654 sini::kan Lab stock FC63 kina::mls (3) HV1326 kinb::cm Lab stock HV1260 kinc::cm (4) DL153 kind::tet (5) HV1372 kine::cm This study ALM105 kinc::mls kind::tet (3) SSB46 srfaa::mls (2) PB214 kina::mls amye::p hyperc101 -yfp (spec) This study PB224 kinb::cm amye::p hyperc101 -yfp (spec) This study PB225 kinc::cm amye::p hyperc101 -yfp (spec) This study PB166 kind::mls amye::p hyperc101 -yfp (spec) This study PB215 kine::cm amye::p hyperc101 -yfp (spec) This study PB227 kinc::mls kind::tet amye::p hyperc101 -yfp(spec) This study PB283 srfaa::mls amye::p hyperc101 -yfp (spec) This study YC704 gale::tet (6) YC534 galkt::kan (6) YC705 gale::tet galkt::kan (6) PB384 gale::tet amye::p hyperc101 -yfp (spec) This study PB385 galkt::kan amye::p hyperc101 -yfp (spec) This study PB386 gale::tet galkt::kan amye::p hyperc101 -yfp (spec) This study PB08 Bacillus subtilis GB03 Joseph W. Kloepper PB288 Bacillus amyloliquefaciens FZB42 Michael Fischbach Unless indicated, strains are derivatives of B. subtilis Antibiotics: spectinomycin (spec), kanamycin (kan), MLS (mls), chloramphenicol (cm), tetracycline (tet). 1. Vlamakis H, Aguilar C, Losick R, Kolter R (2008) Control of cell fate by the formation of an architecturally complex bacterial community. Genes Dev 22(7): Branda SS, González-Pastor JE, Ben-Yehuda S, Losick R, Kolter R (2001) Fruiting body formation by Bacillus subtilis. Proc Natl Acad Sci USA 98(20): McLoon AL, Guttenplan SB, Kearns DB, Kolter R, Losick R (2011) Tracing the domestication of a biofilm-forming bacterium. J Bacteriol 193(8): Aguilar C, Vlamakis H, Guzman A, Losick R, Kolter R (2010) KinD is a checkpoint protein linking spore formation to extracellular-matrix production in Bacillus subtilis biofilms. MBio 1(1): e López D, Vlamakis H, Losick R, Kolter R (2009) Paracrine signaling in a bacterium. Genes Dev 23(14): Chai Y, Beauregard PB, Vlamakis H, Losick R, Kolter R (2012) Galactose metabolism plays a crucial role in biofilm formation by Bacillus subtilis. MBio 3(4):e of6