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Fujita and Losick
Supplemental Material
Plasmid construction
In what follows, Phyper-spank is abbreviated as Phy-spank (Britton et al. 2002). All plasmid constructions were performed with Escherichia coli DH5a by using standard methods. The plasmid (pMF274) used to generate spo0AWPhy-spank-spo0A spc (MF2001) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of Spo0A (oligonucleotide primers omf288 and omf289 and template DNA B. subtilis PY79), and pPE30 (Britton et al. 2002) cut with SalI-SphI. The plasmid (pMF299) used to generate spo0AWPhy-spank-spo0A* spc (MF2146) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of spo0A* (oligonucleotide primers omf288 and omf289 and template DNA pMF113 (Fujita and Losick 2003) and pPE30 cut with SalI-SphI. The plasmid (pMF262) used to generate kinAWPhy-spank-kinA spc (MF1887) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of KinA (oligonucleotide primers omf246 and omf247 and template DNA B. subtilis PY79), and pPE30 cut with SalI-SphI. The plasmid (pMF263) used to generate kinBWPhy-spank-kinB spc (MF1888) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of KinB (oligonucleotide primers omf248 and omf249 and template DNA B. subtilis PY79), and pPE30 cut with SalI-SphI. The plasmid (pMF264) used to generate kinCWPhy-spank-kinC spc (MF1889) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of KinC (oligonucleotide primers omf250 and omf251 and template DNA B. subtilis PY79), and pPE30 cut with SalI-SphI. The plasmid (pMF300) used to generate kinDWPhy-spank-kinD spc (MF2147) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of KinD (oligonucleotide primers omf301 and omf302 and template DNA B. subtilis PY79), and pPE30 cut with SalI-SphI. The plasmid (pMF301) used to generate kinEWPhy-spank-kinE spc (MF2148) was created by ligating the SalI-SphI PCR fragment containing amino-terminus portion of KinE (oligonucleotide primers omf303 and omf304 and template DNA B. subtilis PY79), and pPE30 cut with SalI-SphI. The plasmid (pMF305) used to generate amyE:: Phy-spank-spo0A* kan (MF2251) was created by ligating the SalI-SphI PCR fragment containing Spo0A* (oligonucleotide primers omf288 and omf318 and template DNA pMF113, and pDP111 (the gift of D. Kearns) cut with SalI-SphI. The plasmid (pMF138) used to generate kinA::kinA-gfp spc (MF928) was created by ligating the EcoRI-XhoI PCR fragment containing carboxy-teminus of KinA (oligonucleotide primers omf99 and omf100 and template DNA B. subtilis PY79), and pKL147 (Lemon and Grossman 1998) cut with EcoRI-XhoI. The plasmid (pMF139) used to generate kinA::kinA-gfp kan (MF929) was created by ligating the EcoRI-XhoI PCR fragment containing carboxy-teminus of KinA (oligonucleotide primers omf99 and omf100 and template DNA B. subtilis PY79), and pKL168 (Lemon and Grossman 1998) cut with EcoRI-XhoI. The plasmid (pMF13) used to generate rpoCWrpoC–gfp spc (MF60) was created by ligating the EcoRI-XhoI PCR fragment containing 3’ end of rpoC (oligonucleotide primers omf8 and 9 and template DNA B. subtilis PY79), and pKL147 cut with EcoRI and XhoI. The plasmid (pMF131) used to generate amyE::PspoIID-lacZ spc (MF873) was created by ligating the EcoRI-HindIII PCR fragment containing promoter region of spoIID (oligonucleotide primers omf37 and 38 and template DNA B. subtilis PY79), and pDG1728 cut with EcoRI-HindIII. The plasmid (pEG180) used to generate amyE::PgerE-gfp (EG180) was created by ligating the EcoRI-HindIII PCR fragment containing promoter region of gerE (oligonucleotide primers gerE-up and gerE-low, and template DNA B. subtilis PY79), and the large fragment of pMF35 (Fujita and Losick 2002) cut with EcoRI-HindIII (constructed by J.E. Gonzalez-Pastor). The plasmid (pMF303) used to generate amyE::PgerE-lacZ (MF2240) was created by ligating the EcoRI-HindIII fragment containing promoter region of gerE and pDG1728 cut with EcoRI-HindIII. Oligonucleotide primers used for this study are listed in supplemental-Table 2. The plasmid pDAG32 was used to switch spc for cm (Fujita et al. 2005).
Supplemental Figure S1.
Chromosome remodeling in a kinA-inducible strain.
Cells of strain MF2014 (Phy-spank-kinA, rpoC-gfp) were induced to sporulate by the addition of 20 mM IPTG in CH medium. Typical examples of nucleoids in uninduced cells (-IPTG) are shown in the left-hand panels. Typical axial filaments in induced cells (+IPTG) are shown in the right-hand panels. Gaps between cell poles and the edge of nucleoids are indicated by the arrows in the left-hand panels. Close opposition of the ends of the axial filaments with the cells poles are indicated by the arrows in the right-hand panels. Membrane and DNA were visualized by FM 4-64 and RNA polymerase-GFP, respectively. The scale bar corresponds to 2 mm.
Supplemental Figure S2.
Relative timing of transcription from low- and high-threshold promoters as monitored by use of lacZ fusions.
(A) β-galactosidase levels were measured in strains MF1248 (Pskf-lacZ, open circles) and MF290 (PspoIIG-lacZ, open triangles). Samples were collected at the indicated times following suspension in the SM medium and assayed for β-galactosidase activity. For each experiment, the specific activities (Miller units) were normalized to the maximum activity (typically, 50 Miller units for skf, and 100 Miller units for spoIIG).
(B) β-galactosidase levels were measured in strains MF2205 (Phy-spank-kinA, Pskf-lacZ filled circles), and MF2206 (Phy-spank-spo0A*, Pskf-lacZ, gray circles). Samples were collected at the indicated times following IPTG addition in CH medium and assayed for β-galactosidase activity. The specific activities (Miller units) were normalized to the maximum activity as indicated in (A).
(C) β-galactosidase levels were measured in strains MF1917 (Phy-spank-kinA, PspoIIG-lacZ, filled triangle), and MF2176 (Phy-spank-spo0A*, PspoIIG-lacZ, gray triangles). Samples were collected and assayed as in (B).
Supplemental Figure S3.
Induction of KinA synthesis triggers sporulation in a CodY-dependent manner in CH medium supplemented with glucose.
Accumulation of β-galactosidase was measured in strains harboring lacZ fused to the spoIIG promoter in a KinA-inducible strain in medium supplemented with glucose. Cells of strain MF1917 (Phy-spank-kinA, PspoIIG-lacZ) were induced to sporulate by the addition of IPTG in CH medium with (filled diamonds) and without (open diamonds) glucose (0.2%). Cells of strain MF2367 (codYD, Phy-spank-kinA, PspoIIG-lacZ) were induced to sporulate by the addition of IPTG to CH medium supplemented with glucose (0.2%) (gray diamonds). Also shown are the results with cells of strain MF290 (PspoIIG-lacZ), which is not inducible for kinA (that is, lacks Phy-spank-kinA), in CH medium with (filled squares) and without (open squares) glucose.
Supplemental TABLE S1
Bacterial strains and plasmids used for this study
Strain Genotype Reference/source/construction
PY79 prototroph (Youngman et al. 1983)
PE128 amyE::PspoIIQ-gfp spc (Eichenberger et al. 2001)
EG180 amyE::PgerE-gfp spc pEG180 à PY79
EG297 skfWPskf-skfA'-gfp spc (Gonzalez-Pastor et al. 2003)
MF60 rpoCWrpoC-gfp spc pMF13 à PY79
MF237 amyE::PspoIIG-gfp spc (Fujita and Losick 2002)
MF248 amyE::PspoIID-gfp spc (Fujita and Losick 2003)
MF290 amyE::PspoIIG-lacZ spc (Fujita and Losick 2002)
MF873 amyE::PspoIID-lacZ spc pMF131 à PY79
MF928 kinA::kinA-gfp spc pMF138 à PY79
MF929 kinA::kinA-gfp kan pMF139 à PY79
MF1248 amyE::Pskf-lacZ spc (Fujita et al. 2005)
MF1887 kinAWPhy-spank-kinA spc pMF262 à PY79
MF1888 kinBWPhy-spank-kinB spc pMF263 à PY79
MF1889 kinCWPhy-spank-kinC spc pMF264 à PY79
MF1913 kinAWPhy-spank-kinA cm pDAG32 à MF1889
MF1917 kinAWPhy-spank-kinA cm, amyE::PspoIIG-lacZ spc MF290 à MF1913
MF1929 kinAWPhy-spank-kinA cm, amyE::PspoIIG-gfp spc MF237 à MF1913
MF1956 kinAWPhy-spank-kinA cm, amyE::PspoIIQ-gfp spc PE128 à MF1913
MF1957 kinAWPhy-spank-kinA cm, amyE::PspoIID-gfp spc MF248 à MF1913
MF1959 kinAWPhy-spank-kinA cm, skfWPskf-skfA'-gfp spc EG297 à MF1913
MF1996 kinAWPhy-spank-kinA-gfp spc kan pMF262 à MF928
MF2001 spo0AWPhy-spank-spo0A spc pMF274 à PY79
MF2008 spo0AWPhy-spank-spo0A cm pDAG32 à MF2001
MF2014 kinAWPhy-spank-kinA cm, rpoCWrpoC-gfp spc MF60 à MF1913
MF2035 kinAWPhy-spank-kinA cm, amyE::PgerE-gfp spc EG180 à MF1913
MF2146 spo0AWPhy-spank-spo0A* spc pMF299 à PY79
MF2147 kinDWPhy-spank-kinD spc pMF300 à PY79
MF2148 kinEWPhy-spank-kinE spc pMF301 à PY79
MF2158 spo0AWPhy-spank-spo0A* cm pDAG32 à MF2146
MF2175 spo0AWPhy-spank-spo0A cm, amyE::PspoIIG-lacZ spc MF290 à MF2008
MF2176 spo0AWPhy-spank-spo0A* cm, amyE::PspoIIG-lacZ spc MF290 à MF2158
MF2177 spo0AWPhy-spank-spo0A* cm, amyE::PspoIIG-gfp spc MF237 à MF2158
MF2202 spo0AWPhy-spank-spo0A* cm, skfWPskf-gfp spc EG297 à MF2158
MF2205 kinAWPhy-spank-kinA cm, amyE::Pskf-lacZ spc MF1248 à MF1913
MF2206 spo0AWPhy-spank-spo0A* cm, amyE::Pskf-lacZ spc MF1248 à MF2158
MF2210 kinAWPhy-spank-kinA cm, amyE::PspoIID-lacZ spc MF873 à MF1913
MF2238 spo0AWPhy-spank-spo0A cm, amyE::PgerE-gfp spc EG180 à MF2008
MF2239 spo0AWPhy-spank-spo0A* cm, amyE::PgerE-gfp spc EG180 à MF2158
MF2240 amyE::PgerE-lacZ spc pMF303 à PY79
MF2242 kinAWPhy-spank-kinA cm, amyE::PgerE-lacZ spc MF2240 à MF1913
MF2243 spo0AWPhy-spank-spo0A cm, amyE::PgerE-lacZ spc MF2240 à MF2008
MF2244 spo0AWPhy-spank-spo0A* cm, amyE::PgerE-lacZ spc MF2240 à MF2158
MF2251 amyE::Phy-spank-spo0A* kan pMF305 à PY79
MF2252 kinAWPhy-spank-kinA cm, amyE::Phy-spank-spo0A* kan MF2251 à MF1913
MF2367 kinAWPhy-spank-kinA cm, amyE::PspoIIG-lacZ spc, codYD::erm PS258 (gift of A. L. Sonenshein) à MF1917
Supplemental TABLE S2
Sequence of oligonucleotide primers used
Oligo Sequence
omf8 5’-gccGAATTCgccaagtactgacagga-3’
omf9 5’-gccCTCGAGttcaaccgggaccatatcgtcagtc-3’
omf37 5'-gcgGAATTCgcagacatcgaacgtgtaaat-3’
omf38 5'-gcgAAGCTTctgctcgggattcgactcta-3’
omf99 5'-aggGAATTCgacaatggaaggcaacgaacattac-3’
omf100 5'-aggCTCGAGtttttttggssstgsssttttssscgc-3’
omf246 5'-gccGTCGACacataaggaggaactactatggaacaggatacgcagcatgtt-3’
omf247 5'-gccGCATGCgctgtttacatagacgatcttgcc-3’
omf248 5'-gccGTCGACacataaggaggaactactatggaaattctaaaagactatctt-3’
omf249 5'-gccGCATGCaagccctgagactgccgcctcgta-3’
omf250 5'-gccGTCGACacataaggaggaactactatgagaaaatatcaagctcgtatc-3’
omf251 5'-gccGCATGCgcctgtaaattggtcatcccggtc-3’
omf288 5'-ggcGTCGACacataaggaggaactactatggagaaaattaaagtttgtgttgc-3’
omf289 5'-gccGCATGCcgatgtcattgtataccattgagat-3’
omf301 5'-gccGTCGACacataaggaggaactactatgttggagcgatgcaaattgaaa-3’
omf302 5'-gccGCATGCatctgtcacattacgttttgagtc-3’
omf303 5'-gccGTCGACacataaggaggaactactatggaaactcttggtgtccagaca-3’
omf304 5'-gccGCATGCaccgccatctttatcaaacaggac-3’
omf318 5'-gccGCATGCttaagaagccttatgctctaacctcagc-3’
gerE-up 5’-acgtcaGAATTCcaagcgcagtcggcagttc-3’
gerE-low 5’- tccagcAAGCTTcttcgattgaaattctttctccttc-3’
Restriction endonuclease sites are in capital letters.
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