Supplemental materials for Evidence for cyclic-di-gmp-mediated signaling pathway in Bacillus subtilis by Chen Y. et al. 1. Table S1. Strains used in this study 2. Table S2. Plasmids used in this study 3. Table S1. Primers used in this study 4. Supplemental methods 5. Supplemental references
Table S1. Strains used in this study. strain genotype reference E. coli XL1 Blue an E. coli strain used for molecular cloning Invitrogen BTH101 an E. coli host strain for bacterial two hybrid assay (6) RL219 RL1927 RL1936 DH5α derivative containing the plasmid pdg268, Amp R, Cm R DH5α derivative containing the plasmid pdg1662, Amp R, Spc R DH5α derivative containing the plasmid pdg780, Amp R, Kan R (1) (5) Losick lab collection RL3002 RL3544 DH5α derivative containing the plasmid pdr111, Amp R, Spc R DH5α derivative containing the plasmid pah54, Amp R, Spc R (7) Losick lab collection RL3545 DH5α derivative containing the plasmid pah52, Amp R, Mls R Losick lab collection RL4505 DH5α derivative containing the plasmid pac225, Amp R, Cm R Losick lab collection B. subtilis PY79 laboratory strain used as a host for transformation 3610 undomesticated wild strain capable of forming robust biofilms (2) RL4620 spo0a in 3610, Kan R Losick lab collection RL4573 kina kinb in 3610, Mls R, Kan R (8) RL5273 kinc kind in 3610, Mls R, Tet R (8) YC110 amye::p epsa -lacz in 3610, Cm R (3) YC121 amye::p tapa -lacz in 3610, Cm R (4) CY3 ypfa in 3610, Kan R this study CY5 yhck in 3610, Mls R this study CY7 ytrp in 3610, Spec R this study CY8 yybt in 3610, Kan R this study CY9 yuxh in 3610, Mls R this study CY10 ykow in 3610, Mls R this study CY11 ykui in 3610, Cm R this study CY24 yybt yhck ytrp in 3610, Kan R, Mls R, Spec R this study CY25 yuxh ykui ykow in 3610, Spec R, Cm R, Mls R this study CY30 yuxh ypfa in 3610, Mls R, Kan R this study CY84 amye::p hyspank -ypfa in 3610, Spec R this study CY85 yuxh, amye::p hyspank -ypfa in 3610, Mls R, Spec R this study CY87 yuxh, amye::yuxh wt in 3610, Mls R, Cm R this study CY88 yuxh, amye::yuxh mut (ELL>AEL) in 3610, Mls R, Cm R this study CY89 yuxh, amye::yuxh mut (ELL>EDA) in 3610, Mls R, Cm R this study CY110 amye::p yuxh -lacz in 3610, Cm R this study CY121 spo0a, amye::p yuxh -lacz in 3610, Kan R, Cm R this study CY158 yuxh, ypfa, amye::p hyspank -gst-ypfa in 3610, Mls R, Kan R, Spec R this study CY182 yuxh, ypfa, amye::p hyspank -gst in 3610, Mls R, Kan R, Spec R this study CY230 kina kinb ypfa in 3610, Cm R, Mls R, Kan R this study CY231 kinc kind ypfa in 3610, Mls R, Tet R, Kan R this study CY301 ypfa-pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY302 ypfa-pknt25, flig-pch363 in BTH101, Kan R, Amp R this study CY303 yabk-pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY304 yabk-pknt25, flig-pch363 in BTH101, Kan R, Amp R this study CY305 ypfa-pknt25, pch363 in BTH101, Kan R, Amp R this study CY306 yabk-pknt25, pch363 in BTH101, Kan R, Amp R this study CY307 pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY308 pknt25, flig-pch363 in BTH101, Kan R, Amp R this study CY309 pknt25, pch363 in BTH101, Kan R, Amp R this study CY331 yuxh, amye::p hyspank -ypfa R100F in 3610, Mls R, Spec R this study CY332 yuxh, amye::p hyspank ypfa R104D in 3610, Mls R, Spec R this study CY357 ypfa R100F -pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY358 ypfa R104D -pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY372 ypfa K24D -pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY373 ypfa N127A -pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY375 ypfa G132A -pknt25, mota-pch363 in BTH101, Kan R, Amp R this study CY396 yuxh, amye::p hyspank -ypfa K24D in 3610, Mls R, Spec R this study CY397 yuxh, amye::p hyspank -ypfa N127A in 3610, Mls R, Spec R this study CY399 yuxh, amye::p hyspank -ypfa G132A in 3610, Mls R, Spec R this study CY437 ypfa, amye::p epsa -lacz in 3610, Kan R,Cm R this study CY438 ypfa, amye:: P tapa -lacz in 3610, Kan R,Cm R this study
Table 2. Plasmids used in this study. pah52 a plasmid for the LHF PCR drug template, Amp R, MLS R Losick lab collection pah54 a pbskii (+) derivative as template for the LHF PCR mutagenesis, Amp R, Spec R Losick lab collection pdg268 pdg780 an amye integration vector that contains a promoter-less lacz, Cm R, Amp R a plasmid for Campbell integration in B. subtilis, Kan R, Amp R (1) Losick lab collection pdg1662 a plasmid for integration at amye in B. subtilis, Cm R, Amp R (5) pdr111 pch363 pknt25 an amye integration vector that contains P hyspank, Spec R, Amp R T18 adenylate cylcase domain, Amp R T25 adenylate cyclase domain, Kan R (7) (6) (6) pgex-2tk an expression vector for GST fusion proteins GE Healthcare pcy58 amye::yuxh mut ( 88 ELL 90 > 88 AEL 90 ) in pdg1662 this study pcy59 amye::yuxh mut ( 88 ELL 90 > 88 EDA 90 ) in pdg1662 this study pcy60 amye::yuxh wt in pdg1662 this study pcy83 amye::p hyspank -ypfa in pdr111 this study pcy88 amye::p hyspank -gst in pdr111 this study pcy100 amye::p yuxh -lacz in pdg268 this study pcy152 amye::p hyspank- gst-ypfa in pdr111 this study pcy283 ypfa cloned into pknt25 between BamHI and EcoRI this study pcy301 yabk cloned into pknt25 between BamHI and EcoRI this study pcy302 mota cloned into pch363 between BamHI and EcoRI this study pcy303 flig cloned into pch363 between KpnI and EcoRI this study
Table 3. Primers used in this study. yhck-ko-p1 yhck-ko-p2 yhck-ko-p3 yhck-ko-p4 ytrp-ko-p1 ytrp-ko-p2 ytrp-ko-p3 ytrp-ko-p4 ypfa-ko-p1 ypfa-ko-p2 ypfa-ko-p3 ypfa KO-P4 yybt-ko-p1 yybt-ko-p2 yybt-ko-p3 yybt-ko-p4 yuxh-ko-p1 yuxh-ko-p2 yuxh-ko-p3 yuxh-ko-p4 P yuxh -F1 P yuxh -R1 yuxh-m1-f yuxh-m1-r yuxh-m2-f yuxh-m2-r yuxh-r1 ypfa-f ypfa-r pgex-ypfa-f1 pgex-ypfa-r1 gst-ypfa-f3 gst-ypfa-r2 gst-ck-f1 gst-ck-r3 ypfa-mut K24D -F ypfa-mut K24D -R ypfa-mut R100F -F ypfa-mut R100F -R ypfa-mut R104D -F ypfa-mut R104D- R ypfa-mut N127A -F ypfa-mut N127A -R ypfa-mut G132V -F ypfa-mut G132V -R B 2 ypfa-f B 2 ypfa-r1 B 2 mota-f B 2 mota-r B 2 flig-f B 2 flig-r1 B 2 yabk-f B 2 yabk-r 5 -ATAAACATAGTGATTAACGCGGC-3 5 -CAATTCGCCCTATAGTGAGTCGTCAGTTCTTTCAGCAAATATT-3 5 -CCAGCTTTTGTTCCCTTTAGTGAGAAGAATGAATTCAATGTTCGA-3 5 -GATAAGTAATAGGAATTGACAAC-3 5 -GATCATTGGGAGCAGCAGGCGCA-3 5 -CAATTCGCCCTATAGTGAGTCGTTTAGTTTGTTCTACCATGTT-3 5 -CCAGCTTTTGTTCCCTTTAGTGAGGCTTGATGATTCATGACTCA-3 5 -TAATGTGGAGTCAATTGTGACTT-3 5 -ACCTTCGCTGGATGGACGTAGAA-3 5 -CAATTCGCCCTATAGTGAGTCGTTCTCCAATCTCTATCATTGAC-3 5 -CCAGCTTTTGTTCCCTTTAGTGAGCGAATGGAATAAACATCGGC-3 5 -GTGGCATTTGCCATCCTCAGCGGG-3 5 -ATTCGTATGTTCAGGATGATACG-3 5 -CAATTCGCCCTATAGTGAGTCGTAGCTTGGCATTTCTATCACTC-3 5 -CCAGCTTTTGTTCCCTTTAGTGAGGCGTACAGAGATGAAGGTTA-3 5 -CTTGTGCGTTCTGCTTCTCACAC-3 5 -ATGCGCATGCGCTTGTGCAGGCTT-3 5 -CAATTCGCCCTATAGTGAGTCGT CACCCTCATTGATTTATC-3 5 -CCAGCTTTTGTTCCCTTTAGTGAGGACGCAAAATGATTGCGG-3 5 -CATTTATTGTATCGGTAGAAC-3 5 -GTACGAATTCGAAGAAGAGCTTGAGGAAGAA-3 5 -GTACGGATCCTTACAATACGCGCGGCTTTCA-3 5 -TGTTGCTTATGCAGAGCTGTATAGAGAAA-3 5 -TTTCTCTATACAGCTCTGCATAAGCAACA-3 5 -TTGTCATTGAAGACGCTGAAGATATAC-3 5 -GTATATCTTCAGCGTCTTCAATGACAA-3 5 -GTCAGGATCCTCATTTTGCGTCCATAAGATT-3 5 -GTACAAGCTTAAGGAGGAACTACTATGATAGAGATTGGAGAAAAT-3 5 -GACTGCTAGCTTATTCCATTCGGGCCTTTCT-3 5 -GTACGGATCCATGATAGAGATTGGAGAAAAT-3 5 -GTACGAATTCTTATTCCATTCGGGCCTTTCT-3 5 -GTACAAGCTTAAGGAGGAACTACTATGTCCCCTATACTAGGTTATTG-3 5 -GTACGCTAGCTTATTCCATTCGGGCCTTTCT-3 5 -GTACAAGCTTAAGGAGGAACTACTATGTCCCCTATACTAGGTTAT-3 5 -GTACGCTAGCTCAAACAGATGCACGACGAG-3 5 -TGAATTGAAAAAGGCAAAAAGCGATGCGGTCAGCATCGAAAACAATG-3 5 -CATTGTTTTCGATGCGACCGCATCGCTTTTTGCCTTTTTCAATTCA-3 5 -AAAATGAAAAGAATCCAGCGCTTCCAATATGTAAGAACTGATGCG-3 5 -CGCATCAGTTCTTACATATTGGAAGCGCTGGATTCTTTCATTTT-3 5 -ATCCAGCGCCGCCAATATGTAGACACTGATGCGGTATTAGATGTG-3 5 -CACATCTAATACCGCATCAGTGTCTACATATTGGCGGCGCTGGAT-3 5 -GAGATCCGCACACTATCCTATCTCATCAGTGCAGGCGGCATCGCC-3 5 -GGCGATGCCGCCTGCACTGATGAGATAGGATAGTGTGCGGATCTC-3 5 -ATCCTATAACATCAGTGCAGGCGTCATCGCCGTGGTTTTAGCTGATG-3 5 -CATCAGCTAAAACCACGGCGATGACGCCTGCACTGATGTTATAGGAT-3 5 -GTACGGATCCGATAGAGATTGGAGAAAATGTA-3 5 -GTACGAATTCCGTTCCATTCGGGCCTTTCTTCT-3 5 -GTACGGATCCGATGGATAAAACTTCGTTAATCG-3 5 -GTACGAATTCCGTGCTTCTTCTTCTTTTTTCTCGC-3 5 -GTACGGTACCGATGGCGAGACGTGATCAAGATAAG-3 5 -GTACGAATTCCGGACAATAATATCATCCCCTC-3 5 -GTACGGATCCGATGGCTTTGCATTATTATTGTC-3 5 -GTACGAATTCCGTTGAATAAATGTGTGATATTC-3
Supplemental methods Assays of swimming motility. The effect on swimming motility by overexpression of YpfA in B. subtilis was examined as follows. CY85 ( yuxh amye::p hyspank -ypfa) cells were grown in LB shaking culture to O.D. 600 about 0.3. IPTG was added to the culture at a final concentration of 50 µm. After 30 min of incubation at 37 C, 50 µl of cell suspension was quickly spotted on a cover slide and analyzed by time-lapse phasecontrast microscopy. 3610 cells treated with IPTG and CY85 cells without IPTG treatment were used as controls. Cell movement in each sample was recorded for about 10 seconds.
Supplemental references 1. Antoniewski, C., B. Savelli, and P. Stragier. 1990. The spoiij gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes. J. Bacteriol. 172:86-93. 2. Branda, S. S., J. E. Gonzalez-Pastor, S. Ben-Yehuda, R. Losick, and R. Kolter. 2001. Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci. USA 98:11621-11626. 3. Chai, Y., F. Chu, R. Kolter, and R. Losick. 2008. Bistability and biofilm formation in Bacillus subtilis. Mol. Microbiol. 67:254-263. 4. Chai, Y., R. Kolter, and R. Losick. 2009. Paralogous antirepressors acting on the master regulator for biofilm formation in Bacillus subtilis. Molecular Microbiology 74:876-887. 5. Guérout-Fleury, A. M., N. Frandsen, and P. Stragier. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57-61. 6. Karimova, G., J. Pidoux, A. Ullmann, and D. Ladant. 1998. A bacterial twohybrid system based on a reconstituted signal transduction pathway. Proc. Natl. Acad. Sci. USA 95:5752-5756. 7. Kearns, D. B., and R. Losick. 2005. Cell population heterogeneity during growth of Bacillus subtilis. Genes Dev. 19:3083-3094. 8. McLoon, A. L., I. Kolodkin-Gal, S. M. Rubinstein, R. Kolter, and R. Losick. 2011. Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis. J. Bacteriol. 193:679-685.