Fujita and Losick Supplemental Material

Fujita and Losick

Fujita and Losick Supplemental Material

Plasmid construction

All plasmids constructions were performed in Escherichia coli DH5a using standard methods.

The plasmid used to generate amyE::PspoIIE-gfp spc (pMF23) was created in a three-way ligation with a EcoRI- HindIII PCR fragment containing PspoIIE (oligonucleotide primers omf12 and omf13 and template DNA PY79), a HindIII-BamHI PCR fragment containing gfp (Fujita and Losick 2002), and pDG1730 (Guerout-Fleury et al. 1995) cut with EcoRI-HindIII. The plasmid used to generate amyE::PspoIID-gfp spc (pMF22) was created by ligating the EcoRI-BamHI fragment from pMF15 (Fujita and Losick, 2002) containing PspoIID-gfp into pDG1730 between EcoRI-BamHI. The plasmid used to generate amyE::PspoIIQ-spo0A cm (pMF112) was created in a three-way ligation with a EcoRI-HindIII PCR fragment containing PspoIIQ (oligonucleotide primers omf42 and omf43 and template DNA PY79), a HindIII-BamHI PCR fragment containing spo0A (oligonucleotide primers omf27 and omf28 and template DNA PY79), and pDG1662 (Guerout-Fleury et al. 1995) cut with EcoRI and BamHI. The plasmid used to generate amyE::PspoIIQ-sad67 cm (pMF113) was created in a three-way ligation with a EcoRI-HindIII PCR fragment containing PspoIIQ (as described above), a HindIII-BamHI PCR fragment containing sad67 (oligonucleotide primers omf27 and omf28 and template DNA RL1104), and pDG1662 cut with EcoRI and BamHI. The plasmid used to generate amyE::PspoIID-spo0A cm (pMF114) was created in a three-way ligation with a EcoRI-HindIII PCR fragment containing PspoIID (oligonucleotide primers omf37 and omf38 and template DNA PY79), a HindIII-BamHI PCR fragment containing spo0A (as descrived above), and pDG1662 cut with EcoRI and BamHI. The plasmid used to generate amyE::PspoIID-sad67 cm (pMF115) was created in a three-way ligation with a EcoRI-HindIII PCR fragment containing PspoIID (as descrived above), a HindIII-BamHI PCR fragment containing sad67 (as descrived above), and pDG1662 cut with EcoRI and BamHI. The plasmid used to generate amyE::Pspo0A-spo0A-N cm (pMF154) was created by ligating the EcoRI-BamHI PCR fragment containing Pspo0A-spo0A-N (N-terminus 142 aa of Spo0A with spo0A promoter; oligonucleotide primers omf18 and omf107 and template DNA PY79), and pDG1662 cut with EcoRI and BamHI. The plasmid used to generate amyE::PspoIID-spo0A-N cm (pMF155) was created in a three-way ligation with a EcoRI-HindIII PCR fragment containing PspoIID (as described above), a HindIII-BamHI PCR fragment containing spo0A-N (oligonucleotide primers omf27 and omf107 and template DNA PY79), and pDG1662 cut with EcoRI and BamHI. The plasmid used to generate amyE::PspoIIQ-spo0A-N cm (pMF156) was created in a three-way ligation with a EcoRI-HindIII PCR fragment containing PspoIIQ (as described above), a HindIII-BamHI PCR fragment containing spo0A-N (as described above), and pDG1662 cut with EcoRI and BamHI.

For the expression of phosphorelay proteins in E. coli, over-expression plasmids were constructed as follows. DNA fragment carrying the kinA coding sequence was generated by PCR from chromosomal DNA of PY79 using oligonucleotides (omf151 and omf152) that introduced a NdeI site at the 5’ end and a XhoI site at the 3’ end. The fragment was cloned into pET21b expression vector (Novagen), therefore generating an extension of 6 histidine codons at the 3’ end of the gene (pMF193). DNA fragment carrying the spo0B coding sequence was generated by PCR from chromosomal DNA of PY79 using oligonucleotides (omf126 and omf127) that introduced a NdeI site at the 5’ end and a XhoI site at the 3’ end. The fragment was cloned into pET21b, therefore generating an extension of 6 histidine codons at the 3’ end of the gene (pMF184). DNA fragment carrying the N-terminus phosphorylation domain of Spo0A (Spo0A-N) coding sequence (codons 1 to 142 out of 267 total) was generated by PCR from chromosomal DNA of PY79 using oligonucleotides (omf124 and omf125) that introduced a NdeI site at the 5’ end and a XhoI site at the 3’ end. The fragment was cloned into pET21b, therefore generating an extension of 6 histidine codons at the 3’ end of the gene (pMF182).

References

Guerout-Fleury, A. M., Frandsen, N., and Stragier, P. 1996 Plasmids for ectopic integration in Bacillus subtilis. Gene 180: 57-61.

Fujita, M. and Losick, R. 2002. An investigation into the compartmentalization of the sporulation transcription factor sigmaE in Bacillus subtilis. Mol. Microbiol. 43: 27-38.

Supplementary Table 1.

Supplementary Table 2. Strains used

Strain Genotype/relevant features Source/construction/reference

PY79 prototroph (Youngman et al. 1983)

JDB322 zej-82::Tn917::pTV21D2::pD177.1::pD179.1 kan cm Dworkin (Harvard

University)

PE128 amyE::PspoIIQ-gfp spc (Eichenberger et al., 2001)

RL1104 amyE::PspacIN-sad67 cm, spo0AD::erm lab stock

RL1024 amyE::PspacIN-spo0A cm lab stock

MF237 amyE::PspoIIG-gfp spc (Fujita and Losick, 2002)

MF248 amyE::PspoIID-gfp spc pMF22 g PY79

MF277 amyE::PspoIIE-gfp spc pMF23 g PY79

MF290 amyE::PspoIIG-lacZ spc (Fujita and Losick, 2002)

MF813 zej-82::Tn917::pTV21D2::pD177.1::pD179.1::pCm::Tc kan tet pCm::Tc (Steinmetz and

Richter, 1994)g JDB322

MF825 zej-82::amyE::PspoIIQ-spo0A cm tet pMF112g MF813

MF826 zej-82::amyE::PspoIIQ-sad67 cm tet pMF113g MF813

MF827 zej-82::amyE::PspoIID-spo0A cm tet pMF114g MF813

MF828 zej-82::amyE::PspoIID-sad67 cm tet pMF115g MF813

MF972 zej-82::amyE::Pspo0A-spo0A-N cm tet pMF154g MF813

MF973 zej-82::amyE::PspoIID-spo0A-N cm tet pMF155g MF813

MF974 zej-82::amyE::PspoIIQ-spo0A-N cm tet pMF156g MF813

MF1140 zej-82::amyE::Pspo0A-spo0A-N cm tet , amyE::PspoIIG-lacZ spc MF290 g MF972

MF1144 zej-82::amyE::Pspo0A-spo0A-N cm tet , amyE::PspoIID-gfp spc MF248g MF972

MF1145 zej-82::amyE::Pspo0A-spo0A-N cm tet , amyE::PspoIIQ-gfp spc PE128 g MF972

MF1146 zej-82::amyE::Pspo0A-spo0A-N cm tet , amyE::PspoIIG-gfp spc MF237g MF972

MF1151 zej-82::amyE::PspoIID-spo0A-N cm tet, amyE::PspoIIG-lacZ spc MF290 g MF973

MF1156 zej-82::amyE::PspoIID-spo0A-N cm tet, amyE::PspoIIG-gfp spc MF237 g MF973

MF1157 zej-82::amyE::PspoIID-spo0A-N cm tet, amyE::PspoIID-gfp spc MF248 g MF973

MF1158 zej-82::amyE::PspoIID-spo0A-N cm tet, amyE::PspoIIQ-gfp spc PE128 g MF973

MF1161 zej-82::amyE::PspoIIQ-spo0A-N cm tet, amyE::PspoIIG-lacZ spc MF290 g MF974

MF1166 zej-82::amyE::PspoIIQ-spo0A-N cm tet, amyE::PspoIIG-gfp spc MF237 g MF974

MF1167 zej-82::amyE::PspoIIQ-spo0A-N cm tet, amyE::PspoIID-gfp spc MF248 g MF974

MF1168 zej-82::amyE::PspoIIQ-spo0A-N cm tet, amyE::PspoIIQ-gfp spc PE128g MF680

MF1254 amyE::PspacIN-spo0A spc pCm::Spc (Steinmetz and

Richter, 1994) g RL1024

MF1258 zej-82::amyE::Pspo0A-spo0A-N cm tet, amyE::PspacIN-spo0A spc MF1254 g MF972

References

Eichenberger, P., Fawcett, P., and Losick, R. 2001 A three-protein inhibitor of polar septation during sporulation in Bacillus subtilis. Mol. Microbiol. 42: 1147-1162.

Fujita, M. and Losick, R. 2002. An investigation into the compartmentalization of the sporulation transcription factor sE in Bacillus subtilis. Mol. Microbiol. 43: 27-38.

Steinmetz, M. and Richter, R. 1994 Plasmids designed to alter the antibiotic resistance expressed by insertion mutations in Bacillus subtilis, through in vivo recombination. Gene 142: 79-83.

Youngman, P.J., Perkins, J.B., and Losick, R. 1983. Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917. Proc. Natl. Acad. Sci. 80: 2305-2309.

Supplementary Table 3. Sequence of oligonucleotide primers used

Oligo Sequence

omf12 5' GCCgaattcGATCCCCTCCGCCGCTACCA

omf13 5' GCCaagcttTTATATTCGTTGCCTGTCAT

omf18 5' GCCgaattcATCGATATTTATGGAAAAGA

omf27 5' GCCaagcttACATAAGGAGGAACTACTATGGAGAAAATTAAAGTT

omf28 5' GCCggatccTTAAGAAGCCTTATGCTCTAA

omf37 5' GCGgaattcGCAGACATCGAACGTGTAAAT

omf38 5' GCGaagcttCTGCTCGGGATTCGACTCTA

omf42 5' GCGgaattcCGGTATCGGCTGTTACCATT

omf43 5' CGCaagcttCAGCAACATTCTGAACACTTT

omf107 5' AGTAGTggatccTTAGCGTATAATACTGCTTTGCGATGA

omf124 5' GCCGCcatatgGAGAAAATTAAAGTTTGTGTTGCTGATGA

omf125 5' GCCGCCctcgagAATACTGCTTTGCGATGATGGCGCACGA

omf126 5' GGCGGcatatgAAGGATGTTTCAAAAAATCAAGAAG

omf127 5' GGCGGctcgagGTCCAACCCAATTTCAATCAGACATT

omf151 5' GAAgctagcATGGAACAGGATACGCAGCA

omf152 5' GAActcgagTTTTTTTGGAAATGAAATTTTAAACGC

Restriction endonuclease sites are in lowercase letters.

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